Anti-PD-1 antibodies, antigen-binding portions thereof and checkpoint regulator antogonists comprising the same

ABSTRACT

Checkpoint regulator antagonists that bind specifically to TIGIT, PD-1 and/or PD-L1 are disclosed. Also disclosed are methods of making and using the checkpoint regulator inhibitors, including monospecific, bispecific and trispecific checkpoint regulator antagonists thereof.

This application is a Continuation of U.S. application Ser. No.15/858,963, filed Dec. 29, 2017, which claims priority to U.S.Provisional Patent Application Ser. No. 62/442,642, filed Jan. 5, 2017.The entirety of the aforementioned application is incorporated herein byreference.

FIELD

The present application relates generally to cancer treatment and, inparticular, to checkpoint regulator antagonists, including anti-T cellIg and ITIM domain (TIGIT) inhibitors, PD-1 inhibitors, PD-L1 inhibitorscapable of modulating pathways associated with antitumor immunity,including bispecific and trispecific checkpoint regulator antagoniststhereof.

BACKGROUND

The inability of the host to eliminate cancer cells remains a majorproblem. Although an increasing number of therapeutic monoclonalantibodies have been approved for treatment of various cancers,emergence of resistance to these antibodies is frequently observed,given the many different molecular pathways underlying cancer growth andprogression to metastasis. Although the immune system is the principalmechanism of cancer prevention, cancer cells counteractimmunosurveillance. Natural control mechanisms have been identified thatlimit T-cell activation so as to prevent collateral damage resultingfrom unrestrained T-cell activity. This process has been exploited bytumor cells to evade immune responses. Restoring the capacity of immuneeffector cells, especially T cells, to recognize and eliminate cancer isa major objective in immunotherapy.

The T cell Ig and ITIM domain (TIGIT) protein is an immune regulatorthat can block T cell immunity against cancer cells. TIGIT antagonistsand other immune checkpoint antagonists interfere with (or inhibit) theactivity of an immune checkpoint regulator so that, as a result of thebinding to the checkpoint regulator or its ligand, signaling through thecheckpoint regulator receptor is blocked or inhibited. In addition toTIGIT, other immune checkpoint regulators include the TIGIT ligands,CD112, CD155, PD-1 and its ligands, PD-L1 and PD-L2; CTLA-4 and itsligands, B7-1 and B7-2; TIM-3 and its ligand, Galectin-9; LAG-3 and itsligands, including liver sinusoidal endothelial cell lectin (LSECtin)and Galectin-3; CD122 and its CD122R ligand; B7H3, B and T lymphocyteattenuator (BTLA), and VISTA.

The need exists for improved therapeutic binding antagonists orantibodies and methods of treating cancer and chronic viral infectionswith such reagents. Medicines for use in such improved methods oftreatment may comprise antibodies or antibody fragments thatspecifically bind to TIGIT, PD-1 and/or PD-L1 and reverse or partiallyreverse the TIGIT-, PD-1 and/or PD-L1 mediated suppression of anti-tumoror anti-viral immune responses.

In view of the limitations in the ability of a host to eliminate cancercells, a need exists for more effective compositions and methods forcancer treatment.

SUMMARY

One aspect of the present application relates to an antibody, or anantigen-binding portion thereof, comprising: (1) a heavy chain variableregion, wherein the heavy chain variable region comprises threecomplementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3,wherein HCDR1 has an amino acid sequence that is about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 15,17, 20 and 23, wherein HCDR2 has an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS: 2,4, 7, 9, 12, 13, 16, 18, 21 and 24, and wherein HCDR3 has an amino acidsequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 3, 5, 8, 10, 14, 19, 22 and 25; and (2) alight chain variable region, wherein the light chain variable regioncomprises three complementarity determining regions (LCDRs): LCDR1,LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS:26, 29, 31, 33, 35, 39, 42 and 45, wherein LCDR2 has an amino acidsequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 27, 30, 36, 37, 40, 43 and 46, and whereinLCDR3 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% to about 100% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOS: 28, 32, 34, 38, 41, 44and 47, wherein the antibody, or the antigen-binding portion thereof,binds specifically to human TIGIT.

In some embodiments, the antibody, or the antigen-binding portionthereof, comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% toabout 100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 107, 109, 111, 113, 115, 117, 119, 121, 123and 125; and (2) a light chain variable region having an amino acidsequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 108, 110, 112, 114, 116, 118, 120, 122, 124and 126, wherein the antibody, or the antigen-binding portion thereof,binds specifically to human TIGIT.

Another aspect of the present application relates to an antibody, or anantigen-binding portion thereof, comprising: (1) a heavy chain variableregion, wherein the heavy chain variable region comprises threecomplementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3,wherein HCDR1 has an amino acid sequence that is about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 48, 51, 54,56 and 59, wherein HCDR2 has an amino acid sequence that is about 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to an aminoacid sequence selected from the group consisting of SEQ ID NOS: 49, 52,57 and 60, and wherein HCDR3 has an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS:50, 53, 55, 58 and 61; and (2) a light chain variable region, whereinthe light chain variable region comprises three complementaritydetermining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 hasan amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 62, 65, 68, 69, 70 and 73, wherein LCDR2has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% to about 100% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 63, 66, 71 and 74, and whereinLCDR3 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% to about 100% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOS: 64, 67, 72 and 75,wherein the antibody, or the antigen-binding portion thereof, bindsspecifically to human PD-1.

In some embodiments, the antibody, or the antigen-binding portionthereof, comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% toabout 100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 127, 129, 131, 133, 135 and 137; and (2) alight chain variable region having an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS:128, 130, 132, 134, 136 and 138, wherein the antibody, or theantigen-binding portion thereof, binds specifically to human PD-1.

Another aspect of the present application relates to an antibody, or anantigen-binding portion thereof, comprising: (1) a heavy chain variableregion, wherein the heavy chain variable region comprises threecomplementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3,wherein HCDR1 has an amino acid sequence that is about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 76, 79, 85and 88, wherein HCDR2 has an amino acid sequence that is about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 77, 80, 82,84, 86 and 89, and wherein HCDR3 has an amino acid sequence that isabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous toan amino acid sequence selected from the group consisting of SEQ ID NOS:78, 81, 83, 87 and 90; and (2) a light chain variable region, whereinthe light chain variable region comprises three complementaritydetermining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 hasan amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 91, 94, 98, 101 and 104, wherein LCDR2has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% to about 100% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 92, 95, 99, 102 and 105, andwherein LCDR3 has an amino acid sequence that is about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 93, 96, 97,100, 103 and 106, wherein the antibody, or the antigen-binding portionthereof, binds specifically to human PD-L1.

In some embodiments, the antibody, or the antigen-binding portionthereof, comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% toabout 100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 139, 141, 143, 145, 147, 149, 151 and 153; and(2) a light chain variable region having an amino acid sequence that isabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous toan amino acid sequence selected from the group consisting of SEQ ID NOS:140, 142, 144, 146, 148, 150, 152 and 154, wherein the antibody, or theantigen-binding portion thereof, binds specifically to human PD-L1.

Another aspect of the present application relates to a bispecificantibody, comprising: a first antigen binding domain comprising theantigen-binding portion of the anti-TIGIT, anti-PD-1 and/or anti-PD-L1antibody of the present application; and a second antigen binding domaincomprising the antigen-binding portion of another anti-TIGIT, anti-PD-1antibody and/or anti-PD-L1 antibody of the present application.

In one embodiment, a bispecific checkpoint regulator antagonist,TP-M2T8P5 comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% toabout 100% homologous to the amino acid sequence in SEQ ID NO: 155; and(2) a variable light chain variable region having an amino acid sequencethat is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100%homologous to the amino acid sequence in SEQ ID NO: 156.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M4T8P5 comprises: (1) a light chain variable region 2/heavy chainvariable region 1 (VL2/VH1) having an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to theamino acid sequence in SEQ ID NO: 157; and (2) a heavy chain variable 2region/light chain variable region 1 (VH2/VL1) having an amino acidsequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about100% homologous to the amino acid sequence in SEQ ID NO: 158.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M6T8P5 comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% toabout 100% homologous to the amino acid sequence in SEQ ID NO: 159; (2)a light chain variable region 2/light chain constant region (VL2/CL)having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% to about 100% homologous to the amino acid sequence in SEQID NO: 160; and (3) a heavy chain variable region 1/heavy chain constantregion 1 (VH1/CH1) having an amino acid sequence that is about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to the amino acidsequence in SEQ ID NO: 161.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M6T8L8 comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% toabout 100%/homologous to the amino acid sequence in SEQ ID NO: 162; (2)a light chain variable region 2/light chain constant region (VL2/CL)having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% to about 100% homologous to the amino acid sequence in SEQID NO: 163; and (3) a heavy chain variable region 1/heavy chain constantregion 1 (VH1/CH1) having an amino acid sequence that is about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to the amino acidsequence in SEQ ID NO: 164.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M8T10P1 (TP-83) comprises: (1) a heavy chain variable region havingan amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% to about 100% homologous to the amino acid sequence in SEQ ID NO:165; and (2) a light chain having an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to theamino acid sequence in SEQ ID NO: 166.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M9T10P1 (TP-93) comprises: (1) a heavy chain that is about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to the amino acidsequence in SEQ ID NO: 167; and (2) a light chain having an amino acidsequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about100% homologous to the amino acid sequence in SEQ ID NO: 168.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M9T10P5 comprises: (1) a heavy chain that is about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% to about 100% homologous to the amino acidsequence in SEQ ID NO: 169; and (2) a light chain having an amino acidsequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about100% homologous to the amino acid sequence in SEQ ID NO: 170.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M10T8P5 (TP-92) comprises: (1) a heavy chain that is about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to the amino acidsequence in SEQ ID NO: 171; and (2) a light chain variable region havingan amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% to about 100% homologous to the amino acid sequence in SEQ ID NO:172.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M10T8L8 comprises an amino acid sequence that is about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% to about 100% homologous to the amino acidsequence in SEQ ID NO: 173.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M14T8P5 comprises: (1) a heavy chain variable region 2/heavy chainconstant region 1 (VH2/CH1) having an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to theamino acid sequence in SEQ ID NO: 174; (2) a variable light chain region1/heavy chain constant region 1 (VL1/CH1) having an amino acid sequencethat is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100%homologous to the amino acid sequence in SEQ ID NO: 175; (3) a lightchain variable region 2/light chain constant region (VL2/CL) having anamino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%to about 100% homologous to the amino acid sequence in SEQ ID NO: 176;and (4) a heavy chain variable region 1/light chain constant region(VH1/CL) having an amino acid sequence that is about 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% to about 100% homologous to the amino acid sequencein SEQ ID NO: 177.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M14T8L8 comprises: (1) a heavy chain variable region 2/heavy chainconstant region 1 (VH2/HC1) having an amino acid sequence that is about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% homologous to theamino acid sequence in SEQ ID NO: 178; (2) a variable light chain region1/heavy chain constant region 2 (VL1/HC2) having an amino acid sequencethat is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100%homologous to the amino acid sequence in SEQ ID NO: 179; (3) a lightchain variable region 2/light chain constant region (VL2/CL) having anamino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%to about 100% homologous to the amino acid sequence in SEQ ID NO: 180;and (4) a heavy chain variable region 1/light chain constant region(VH1/CL) having an amino acid sequence that is about 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% to about 100% homologous to the amino acid sequencein SEQ ID NO: 181.

Another aspect of the present application relates to nucleic acidsencoding the antibody, the antigen-binding fragment thereof, or thebispecific antibody of the present application.

Another aspect of the present application relates to expression vectorscomprising the nucleic acids of the present application.

Another aspect of the present application relates to host cellstransformed with the expression vectors of the present application.

Another aspect of the present application relates to a method ofproducing the anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1antibody, antigen-binding fragment thereof, including monospecific,bispecific and trispecific checkpoint regulator anatagonists, andcombinations thereof.

Another aspect of the present application relates to a method ofreducing or depleting regulatory T cells in a tumor of a subject in needthereof, comprising: administering to the subject an effective amountthe antibody, antibody fragment or bispecific checkpoint regulatorantagonist of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of anti-TIGIT mabs competing TIGIT and PVR(CD155), the TIGIT ligand.

FIG. 2 shows that anti-TIGIT mouse mabs enhance human T cell function.

FIGS. 3A-C shows CDR sequences of anti-TIGIT mouse mabs (FIG. 3A);anti-PD-1 mouse mabs (FIG. 3B); and anti-PD-L1 mouse mabs (FIG. 3C).

FIGS. 4A-H shows several embodiments of anti-TIGIT antibody variabledomain sequences (FIGS. 4A-C); anti-PD-1 antibody variable domainsequences (FIGS. 4D-E); and anti-PD-L1 antibody variable domainsequences (FIGS. 4F-H).

FIGS. 5A-G depicts exemplary bispecific checkpoint regulator antagonistsequences against PD1 and TIGIT.

FIG. 6 depicts the binding of anti-TIGIT mouse mabs to cells expressingfull-length human TIGIT (hu TIGIT).

FIG. 7 shows that anti-TIGIT mabs block the interaction between hu TIGITand its ligand, human PVR (CD155) in a cell-based assay.

FIG. 8 shows the enhancement of IFN-γ production from human PBMCs byanti-TIGIT mabs.

FIG. 9 shows the enhancement of primary human T cell proliferation byanti-TIGIT mabs.

FIG. 10A shows binding of anti-TIGIT mabs to hu TIGIT- and cyno-hu TIGITin cell based assays. FIG. 10B shows inhibition of antibody binding tohu TIGIT- and cyno-hu TIGIT by anti-TIGIT mabs in cell based assays.

FIG. 11A shows that anti-PD-1 mabs block the interaction between humanPD-1 and human PD-L1. FIG. 11B shows that anti-PD-1 mabs block theinteraction between cyno hu PD-1 and cyno PD-L1.

FIG. 12A shows the binding of anti-PD-1 mabs to hu PD-L1. FIG. 12B showsthe binding of anti-PD-1 mabs to cyno-hu PD-L1.

FIGS. 13A-C shows the binding affinity of anti-PD-1 antibodies to humanPD-1, including PD-01 mab (FIG. 13A), PD-02 mab (FIG. 13B) and abenchmark (BM) anti-PD-1 mab (FIG. 13C).

FIG. 14 shows the enhancement of IFN-γ production from human PBMCs byanti-PD-1 mabs.

FIG. 15 shows increased IFN-γ production from human PBMCs by acombination of anti-TIGIT- and anti-PD-1 mabs.

FIGS. 16A and 16B show increased proliferation of human T cellproliferation by a combination of anti-TIGIT- and anti-PD-1 mabs.

FIGS. 17A-17N show a variety of different bispecific checkpointregulator antagonists, M1-M14, where: (1) the VH1 and VL1 regionscorrespond to anti-PD1, anti-PD-L1 or any other mab variable domain; and(2) the VH2 and VL2 regions correspond to anti-TIGIT variable domains.

FIGS. 18A-18J show a variety of different trispecific checkpointregulator antagonists, where: (1) the VH1 and VL1 regions correspond toanti-TIGIT variable domains; (2) the VH2 and VL21 regions correspond toanti-PD1, anti-PD-L1 or any other mab variable domain; and (3) thecircular region corresponds to AMG386 or any other biological peptide.

FIGS. 19A-D shows the results of screening assays to identify bispecificantibodies capable of blocking PD-1 binding (FIG. 19A, FIG. 19B) andTIGIT binding (FIG. 19C, FIG. 19D).

FIGS. 20A and 20B shows the ability of bispecific antibodies (TP-83,TP-92 and TP-93) to block binding of PD-L1 to PD-1 (FIG. 20A) and blockbinding of PVR to TIGIT (FIG. 20B).

FIGS. 21A and 21B show Coomasie stained gels showing production ofbispecific antibodies, TP-83, TP-92 and TP-93.

FIG. 22 shows an exemplary size exclusion chromatograph (SEC) profilefor bispecific checkpoint antagonists, TP-93 and TP-83, illustratingmanufacturability of the molecules after one step purification.

FIGS. 23A-D show increased IFN-γ production from human PBMCs (Donor 333,FIG. 23A, FIG. 23C; Donor 287, FIG. 23B, FIG. 23D) with a bispecific mab(TP-93) relative to individual anti-TIGIT (T-08) and anti-PD-1 (PD-01)mabs.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide” includes“one or more” peptides or a “plurality” of such peptides. With respectto the teachings in the present application, any issued patent or patentapplication publication described in this application is expresslyincorporated by reference herein.

As used herein, the term “TIGIT” refers to any form of TIGIT andvariants thereof that retain at least part of the activity of TIGIT.Unless indicated differently, such as by specific reference to humanTIGIT, TIGIT includes all mammalian species of native sequence TIGIT,e.g., human, canine, feline, equine, and bovine. The following is anexemplary human TIGIT amino acid sequence:

(SEQ ID. NO. 182) MTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYECIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYENVLSYRSLGNCSFFTETG 

As used herein, the term “PD-1” refers to any form of PD-1 and variantsthereof that retain at least part of the activity of PD-1. Unlessindicated differently, such as by specific reference to human PD-1, PD-1includes all mammalian species of native sequence PD-1, e.g., human,canine, feline, equine, and bovine. An exemplary human PD-1 amino acidsequences is listed below:

(SEQ ID NO: 183) LDSPDRWNPPTFSPALLVVTEGDNATFTSCFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQ PLRPEDGHCSWPL 

As used herein, the term “PD-L1” refers to any form of PD-L1 andvariants thereof that retain at least part of the activity of PD-L1.Unless indicated differently, such as by specific reference to humanPD-L1, PD-L1 includes all mammalian species of native sequence PD-L1,e.g., human, canine, feline, equine, and bovine. Exemplary human PD-L1amino acid sequences is listed below:

(SEQ ID NO: 184) FTVTVPKDLYVVEYGSNMTIECKEPVEKQLDLAALIVYWEMEDKNIIQEVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLENVTSTLRINTTTNEIFYCTERRLDPEE NHTAELVIPELPLAHPPNER 

The term “agonist” refers to a substance which promotes (i.e., induces,causes, enhances, or increases) the biological activity or effect ofanother molecule. The term agonist encompasses substances which bindreceptor, such as an antibody, and substances which promote receptorfunction without binding thereto (e.g., by activating an associatedprotein).

The term “antagonist” or “inhibitor” refers to a substance thatprevents, blocks, inhibits, neutralizes, or reduces a biologicalactivity or effect of another molecule, such as a receptor.

As used herein, the term “antibody” refers to a polypeptide or apolypeptide complex that specifically recognizes and binds to an antigenthrough one or more immunoglobulin variable regions. An antibody can bea whole antibody, an antigen binding fragment or a single chain thereof.The term “antibody” encompasses various broad classes of polypeptidesthat can be distinguished biochemically. Those skilled in the art willappreciate that heavy chains are classified as alpha, delta, epsilon,gamma, and mu, or α, δ, ε, γ and μ) with some subclasses among them(e.g., γ1-γ4). It is the nature of this chain that determines the“class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are within the scope of thepresent disclosure, the following discussion will generally be directedto the IgG class of immunoglobulin molecules.

Antibodies of the disclosure include, but are not limited to,polyclonal, monoclonal, multispecific, bispecific, trispecific, human,humanized, primatized, chimeric and single chain antibodies. Antibodiesdisclosed herein may be from any animal origin, including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In someembodiments, the variable region may be condricthoid in origin (e.g.,from sharks).

The terms “antibody fragment” or “antigen-binding fragment” are usedwith reference to a portion of an antibody, such as F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library andanti-idiotypic (anti-Id) antibodies. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. The term “antibody fragment” includes aptamers,spiegelmers, and diabodies. The term “antibody fragment” also includesany synthetic or genetically engineered proteins comprisingimmunoglobulin variable regions that act like an antibody by binding toa specific antigen to form a complex.

A “single-chain fragment variable” or “scFv” refers to a fusion proteinof the variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins. In some aspects, the regions are connected with a shortlinker peptide of ten to about 25 amino acids. The linker can be rich inglycine for flexibility, as well as serine or threonine for solubility,and can either connect the N-terminus of the VH with the C-terminus ofthe VL, or vice versa. This protein retains the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker.

With regard to IgGs, a standard immunoglobulin molecule comprises twoidentical light chain polypeptides of molecular weight approximately23,000 Daltons, and two identical heavy chain polypeptides of molecularweight 53,000-70,000. The four chains are typically joined by disulfidebonds in a “Y” configuration where the light chains bracket the heavychains starting at the mouth of the “Y” and continuing through thevariable region.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention, the numbering of the constant region domains in conventionalantibodies increases as they become more distal from the antigen-bindingsite or amino-terminus of the antibody. In conventional antibodies, theN-terminal portion is a variable region and at the C-terminal portion isa constant region; the CH3 and CL domains actually comprise thecarboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VL chains (i.e.CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances,e.g., certain immunoglobulin molecules are derived from camelid speciesor engineered based on camelid immunoglobulins. Alternatively, animmunoglobulin molecule may consist of heavy chains only, with no lightchains or light chains only, with no heavy chains.

In naturally occurring antibodies, the six CDRs present in eachantigen-binding domain are short, non-contiguous sequences of aminoacids that are specifically positioned to form the antigen-bindingdomain as the antibody assumes its three dimensional configuration in anaqueous environment. The remainder of the amino acids in theantigen-binding domains, referred to as “framework” regions, show lessinter-molecular variability. The framework regions largely adopt a.beta.-sheet conformation and the CDRs form loops which connect, and insome cases form part of, the .beta.-sheet structure. Thus, frameworkregions act to form a scaffold that provides for positioning the CDRs incorrect orientation by inter-chain, non-covalent interactions. Theantigen-binding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto its cognate epitope. The amino acids comprising the CDRs and theframework regions, respectively, can be readily identified for any givenheavy or light chain variable region by one of ordinary skill in theart, since they have been precisely defined.

As used herein, the terms “VH1” and “VH2” refer to immunoglobulin heavychain variable domains corresponding to two different bindingspecificities. Likewise, the terms “VL1” and “VL2” refer to light chainvariable domains corresponding to two different binding specificities.When used together, it is to be understood that VH1 and VL1 regionsdefine a common binding specificity and that VH2 and VL2 domains definea second binding specificity

Light chains are classified as either kappa or lambda (K, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

As used herein, the term “light chain constant region” includes aminoacid sequences derived from antibody light chain. Preferably, the lightchain constant region comprises at least one of a constant kappa domainor constant lambda domain.

As used herein, the term “heavy chain constant region” includes aminoacid sequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain constant region comprises at least one of: aCH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region)domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.For example, an antigen-binding polypeptide for use in the disclosuremay comprise a polypeptide chain comprising a CH1 domain; a polypeptidechain comprising a CH1 domain, at least a portion of a hinge domain, anda CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH3 domain, or a polypeptide chain comprising aCH1 domain, at least a portion of a hinge domain, a CH2 domain, and aCH3 domain. In some embodiments, a polypeptide of the disclosurecomprises a polypeptide chain comprising a CH3 domain. Further, anantibody for use in the disclosure may lack at least a portion of a CH2domain (e.g., all or part of a CH2 domain). It should be understood thatthe heavy chain constant region may be modified such that they vary inamino acid sequence from the naturally occurring immunoglobulinmolecule.

The heavy chain constant region of an antibody disclosed herein may bederived from different immunoglobulin molecules. For example, a heavychain constant region of a polypeptide may comprise a CH1 domain derivedfrom an IgG₁ molecule and a hinge region derived from an IgG₃ molecule.In another example, a heavy chain constant region can comprise a hingeregion derived, in part, from an IgG₁ molecule and, in part, from anIgG₃ molecule. In another example, a heavy chain portion can comprise achimeric hinge derived, in part, from an IgG₁ molecule and, in part,from an IgG4 molecule.

A “light chain-heavy chain pair” refers to the collection of a lightchain and heavy chain that can form a dimer through a disulfide bondbetween the CL domain of the light chain and the CH1 domain of the heavychain.

The subunit structures and three dimensional configurations of theconstant regions of the various immunoglobulin classes are well known.As used herein, the term “VH domain” includes the amino terminalvariable domain of an immunoglobulin heavy chain and the term “CH1domain” includes the first (most amino terminal) constant region domainof an immunoglobulin heavy chain. The CH1 domain is adjacent to the VHdomain and is amino terminal to the hinge region of an immunoglobulinheavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system).The CH2 domain is unique in that it is not closely paired with anotherdomain. Rather, two N-linked branched carbohydrate chains are interposedbetween the two CH2 domains of an intact native IgG molecule. The CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains.

As used herein the term “disulfide bond” includes a covalent bond formedbetween two sulfur atoms. The amino acid cysteine comprises a thiolgroup that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, a “variant” of an antibody, an antibody fragment or anantibody domain refers to an antibody, an antibody fragment or anantibody domain that (1) shares a sequence homology of at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% with the original antibody, antibodyfragment or antibody domain, and (2) binds specifically to the sametarget that the original antibody, antibody fragment or antibody domainbinds specifically.

It should be understood that where a sequence homology range ispresented herein, as in e.g., the phrase “about 80% to about 100%”, suchan embodiment includes any and all subranges within, wherein the lowernumber can be any whole number between 80 and 100.

As used herein, the phrase “humanized antibody” refers to an antibodyderived from a non-human antibody, typically a mouse monoclonalantibody. Alternatively, a humanized antibody may be derived from achimeric antibody that retains or substantially retains the antigenbinding properties of the parental, non-human, antibody but whichexhibits diminished immunogenicity as compared to the parental antibodywhen administered to humans.

As used herein, the phrase “chimeric antibody,” refers to an antibodywhere the immunoreactive region or site is obtained or derived from afirst species and the constant region (which may be intact, partial ormodified in accordance with the instant disclosure) is obtained from asecond species. In certain embodiments the target binding region or sitewill be from a non-human source (e.g., mouse or primate) and theconstant region is human.

Included within the scope of the multispecific antibodies of the presentapplication are various compositions and methodologies, includingasymmetric IgG-like antibodies (e.g., triomab/quadroma, TrionPharma/Fresenius Biotech); knobs-into-holes antibodies (Genentech);Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y(Genentech); strand exchange engineered domain (SEED) body (EMD Serono;biolonic, Merus); Fab-exchanged antibodies (Genmab), symmetric IgG-likeantibodies (e.g. dual targeting (DT)-Ig (GSK/Domantis); two-in-oneantibody (Genentech); crosslinked MAbs (Karmanos Cancer Center), mAb²(F-star); Cov X-body (Cov X/Pfizer); dual variable domain (DVD)-Igfusions (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab(Medimmune/AZ); BsAb (ZymoGenetics); HERCULES (Biogen Idec, TvAb,Roche); scFv/Fc fusions; SCORPION (Emergent BioSolutions/Trubion,ZymoGenetics/BMS); dual affinity retargeting technology (Fc-DART),MacroGenics; dual (scFv)₂-Fabs (National Research Center for AntibodyMedicine); F(ab)₂ fusions (Medarex/AMGEN); dual-action or Bis-Fab(Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech);scFv- and diabody-based antibodies (e.g., bispecific T cell engagers(BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DARTs(MacroGenics); single-chain diabodies; TCR-like antibodies (AIT,Receptor Logics); human serum albumin scFv fusion (Merrimack); COMBODIES(Epigen Biotech); and IgG/non-IgG fusions (e.g., immunocytokines(EMDSerono, Philogen, ImmunGene, ImmunoMedics).

By “specifically binds” or “has specificity to”, it is generally meantthat an antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.” In some embodiments, anantibody or an antibody fragment “has specificity to” an antigen if theantibody or antibody fragment forms a complex with the antigen with adissociation constant (K_(d)) of 10⁻⁶M or less, 10⁺⁷M or less, 10⁻⁴M orless, 10⁻⁹M or less, or 10⁻¹⁰M or less.

As used herein, the phrase “chimeric antibody,” refers to an antibodywhere the immunoreactive region or site is obtained or derived from afirst species and the constant region (which may be intact, partial ormodified in accordance with the instant disclosure) is obtained from asecond species. In certain embodiments the target binding region or sitewill be from a non-human source (e.g., mouse or primate) and theconstant region is human.

The term “antagonist antibody” refers to an antibody that binds to atarget and prevents or reduces the biological effect of that target. Insome embodiments, the term can denote an antibody that prevents thetarget, e.g., TIGIT, to which it is bound from performing a biologicalfunction.

As used herein, an “anti-PD-1 antagonist antibody” refers to an antibodythat is able to inhibit PD-1 biological activity and/or downstreamevents(s) mediated by PD-1. Anti-PD-1 antagonist antibodies encompassantibodies that block, antagonize, suppress or reduce (to any degreeincluding significantly) PD-1 biological activity, including downstreamevents mediated by PD-1, such as PD-1 binding and downstream signaling,inhibition of T cell proliferation, inhibition of T cell activation,inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition ofTNF secretion, induction of IL-10, and inhibition of anti-tumor immuneresponses. For purposes of the present invention, it will be explicitlyunderstood that the term “anti-PD-1 antagonist antibody”(interchangeably termed “antagonist PD-1 antibody”, “antagonistanti-PD-1 antibody” or “PD-1 antagonist antibody”) encompasses all thepreviously identified terms, titles, and functional states andcharacteristics whereby PD-1 itself, a PD-1 biological activity, or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree. In some embodiments,an anti-PD-1 antagonist antibody binds PD-1 and upregulates ananti-tumor immune response.

As used herein, an “anti-PD-L1 antagonist antibody” refers to anantibody that is able to inhibit PD-L1 biological activity and/ordownstream events(s) mediated by PD-L1. Anti-PD-L1 antagonist antibodiesencompass antibodies that block, antagonize, suppress or reduce (to anydegree including significantly) PD-L1 biological activity, includingdownstream events mediated by PD-L1, such as PD-L1 binding anddownstream signaling, inhibition of T cell proliferation, inhibition ofT cell activation, inhibition of IFN secretion, inhibition of IL-2secretion, inhibition of TNF secretion, induction of IL-10, andinhibition of anti-tumor immune responses. For purposes of the presentinvention, it will be explicitly understood that the term “anti-PD-L1antagonist antibody” (interchangeably termed “antagonist PD-L1antibody”, “antagonist anti-PD-L1 antibody” or “PD-L1 antagonistantibody”) encompasses all the previously identified terms, titles, andfunctional states and characteristics whereby PD-L1 itself, a PD-L1biological activity, or the consequences of the biological activity, aresubstantially nullified, decreased, or neutralized in any meaningfuldegree. In some embodiments, an anti-PD-L1 antagonist antibody bindsPD-L1 and upregulates an anti-tumor immune response. The phrase “immunecheckpoint regulator” refers to a functional class of agents, whichinhibit or stimulate signaling through an immune checkpoint. An “immunecheckpoint regulator” includes receptors and their associated ligands,which together provide a means for inhibiting or stimulating signalingpathways that otherwise lead to T-cell activation.

The phrases “immune checkpoint binding antagonist” and “immunecheckpoint antagonist” are used interchangeably herein with reference toa class of agents that interfere with (or inhibit) the activity of animmune checkpoint regulator so that, as a result of the binding to thecheckpoint regulator or its ligand, signaling through the checkpointregulator receptor is blocked or inhibited. By inhibiting thissignaling, immune-suppression can be reversed so that T cell immunityagainst cancer cells can be re-established or enhanced. Exemplary immunecheckpoint antagonists include, but are not limited to TIGIT and itsCD155 ligand, PVR; PD-1 and its ligands, PD-L1 and PD-L2; CTLA-4 and itsligands, B7-1 and B7-2; TIM-3 and its ligand, Galectin-9; LAG-3 and itsligands, including liver sinusoidal endothelial cell lectin (LSECtin)and Galectin-3; CD122 and its CD122R ligand; CD70, B7H3, B and Tlymphocyte attenuator (BTLA), and VISTA. Immune checkpoint regulatorantagonists include antibody fragments, peptide inhibitors, dominantnegative peptides and small molecule drugs, either in isolated forms oras part of a fusion protein or conjugate.

The phrases “immune checkpoint binding agonist” and “immune checkpointagonist” are used interchangeably herein with reference to a class ofagents that stimulate the activity of an immune checkpoint regulator sothat, as a result of the binding to the checkpoint regulator or itsligand, signaling through the checkpoint regulator receptor isstimulated. By stimulating this signaling, T cell immunity againstcancer cells can be re-established or enhanced. Exemplary immunecheckpoint regulator agonists include, but are not limited to members ofthe tumor necrosis factor (TNF) receptor superfamily, such as CD27,CD40, OX40 (CD134), glucocorticoid-induced TNFR family-related protein(GITR), and 4-1BB (CD137) and their ligands. Additional checkpointregulator agonists belong to the B7-CD28 superfamily, including CD28 andICOS.

The phrases “dominant-negative protein” or “dominant-negative peptide”refer to a protein or peptide derived from a wild type protein that hasbeen genetically modified by mutation and/or deletion so that themodified protein or peptide interferes with the function of theendogenous wild-type protein from which it is derived.

The phrase “VEGF binding antagonist” refers to a functional class ofagents that bind to VEGF-A or its receptor, VEGFR-2, so that, as aresult of the binding, activation of VEGFR-2 by VEGF-A is blocked orinhibited. As used herein, the term “VEGF binding antagonists” includeantibody fragments, peptide inhibitors, dominant negative peptides andsmall molecule drugs, either in isolated forms or as part of a fusionprotein or conjugate.

The phrase “Tie2 tyrosine kinase receptor binding antagonist” refers toa functional class of agents that bind to a Tie2 tyrosine kinasereceptor or one of its ligands so that, as a result of the binding,activation of the Tie2 tyrosine kinase receptor by one or more of itsligands (i.e., Ang1, Ang2, Ang3 and Ang4) is blocked or inhibited. Asused herein, the term “Tie2 tyrosine kinase receptor binding antagonist”include antibody fragments, peptide inhibitors, dominant negativepeptides and small molecule drugs, either in isolated forms or as partof a fusion protein or conjugate.

The phrase “small molecule drug” refers to a molecular entity, oftenorganic or organometallic, that is not a polymer, that has medicinalactivity, and that has a molecular weight less than about 2 kDa, lessthan about 1 kDa, less than about 900 Da, less than about 800 Da or lessthan about 700 Da. The term encompasses most medicinal compounds termed“drugs” other than protein or nucleic acids, although a small peptide ornucleic acid analog can be considered a small molecule drug. Examplesinclude chemotherapeutic anticancer drugs and enzymatic inhibitors.Small molecules drugs can be derived synthetically, semi-synthetically(i.e., from naturally occurring precursors), or biologically.

As used herein, the term “recombinant” refers to polypeptides orpolynucleotides that do not exist naturally and which may be created bycombining polynucleotides or polypeptides in arrangements that would notnormally occur together. The checkpoint regulator antagonists describedherein are by definition “recombinant.”

When describing polypeptide domain arrangements with hyphens betweenindividual domains (e.g., CH2-CH3), it should be understood that theorder of the listed domains is from the amino terminal end to thecarboxy terminal end.

By “specifically binds” or “has specificity to”, it is generally meantthat an antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

The term “immunoconjugate” refers to an antibody which is fused bycovalent linkage to an inhibitory peptide or small molecule drug. Thepeptide or small molecule drug can be linked to the C-terminus of aconstant heavy chain or to the N-terminus of a variable light and/orheavy chain.

A “linker” may be used to link the peptide or small molecule drug, suchas a maytansinoid, to the checkpoint regulator antagonists in a stable,covalent manner. Linkers can be susceptible to or be substantiallyresistant to acid-induced cleavage, light-induced cleavage,peptidase-induced cleavage, esterase-induced cleavage, and disulfidebond cleavage, at conditions under which the compound or the antibodyremains active. Suitable linkers are well known in the art and include,for example, disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups and esterase labile groups.Linkers also include charged linkers, and hydrophilic forms thereof asdescribed herein and know in the art. The immunoconjugate may furtherinclude a flexible 3-15 amino acid peptide (or spacer) between acheckpoint regulator antagonist and the peptide and/or small moleculedrug.

As used herein, the phrase “multispecific inhibitor” refers to amolecule comprising at least two targeting domains with differentbinding specificities. In some embodiments, the multispecific inhibitoris an polypeptide comprising a scaffold and two or more immunoglobulinantigen binding domains targeting different antigens or epitopes. Incertain embodiments, the multispecific inhibitor is a bispecificantibody. In certain embodiments, the multispecific inhibitor is atrispecific antibody.

As used herein, the phrase “bispecific checkpoint regulator antagonist”refers to a molecule comprising at least two targeting domains withdifferent binding specificities. Each targeting domain is capable ofbinding specifically to a target molecule and inhibiting a biologicalfunction of the target molecule upon binding to the target molecule. Insome embodiments, the bispecific checkpoint regulator antagonist is apolymeric molecule having two or more peptides. In some embodiments, thetargeting domain comprises an antigen binding domain or a CDR of anantibody. In some embodiments, the bispecific inhibitor is a bispecificantibody.

The terms “bispecific antibody” and “bispecific checkpoint regulatorantagonist” are used interchangeably herein with reference to anantibody that can specifically bind two different antigens (orepitopes). In some embodiments, the bispecific antibody is a full-lengthantibody that binds one antigen (or epitope) on one of its two bindingarms (one pair of HC/LC), and binds a different antigen (or epitope) onits second arm (a different pair of HC/LC). In these embodiments, thebispecific antibody has two distinct antigen binding arms (in bothspecificity and CDR sequences), and is monovalent for each antigen itbinds to.

In other embodiments, the bispecific antibody is a full-length antibodythat can bind two different antigens (or epitopes) in each of its twobinding arms (two pairs of HC/LC) In these embodiments, the bispecificantibody has two identical antigen binding arms, with identicalspecificity and identical CDR sequences, and is bivalent for eachantigen it binds to.

The terms “bispecific antibody” and “bispecific checkpoint regulatorantagonist” are used interchangeably herein with reference to a moleculecomprising three targeting domains with three different bindingspecificities. Each targeting domain is capable of binding specificallyto a target molecule and inhibiting a biological function of the targetmolecule upon binding to the target molecule. In some embodiments, thetrispecific checkpoint regulator antagonist is a polymeric moleculehaving two or more peptides. In some embodiments, the targeting domaincomprises an antigen binding domain or a CDR of an antibody. In someembodiments, the trispecific checkpoint regulator antagonist is atrispecific antibody.

Exemplary bispecific and trispecific antibodies may include asymmetricIgG-like antibodies (e.g., triomab/quadroma, Trion Pharma/FreseniusBiotech); knobs-into-holes antibodies (Genentech); Cross MAbs (Roche);electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strandexchange engineered domain (SEED) body (EMD Serono; biolonic, Merus);Fab-exchanged antibodies (Genmab), symmetric IgG-like antibodies (e.g.dual targeting (DT)-Ig (GSK/Domantis); two-in-one antibody (Genentech);crosslinked MAbs (Karmanos Cancer Center), mAb² (F-star); Cov X-body(Cov X/Pfizer); dual variable domain (DVD)-Ig fusions (Abbott); IgG-likebispecific antibodies (Eli Lilly); Ts2Ab (Medimmune/AZ); BsAb(ZymoGenetics); HERCULES (Biogen Idec, TvAb, Roche); scFv/Fc fusions;SCORPION (Emergent BioSolutions/Trubion, ZymoGenetics/BMS); dualaffinity retargeting technology (Fc-DART), MacroGenics; dual(scFv)₂-Fabs (National Research Center for Antibody Medicine); F(ab)₂fusions (Medarex/AMGEN); dual-action or Bis-Fab (Genentech);Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv- anddiabody-based antibodies (e.g., bispecific T cell engagers (BiTEs,Micromet); tandem diabodies (Tandab, Affimed); DARTs (MacroGenics);single-chain diabodies; TCR-like antibodies (AIT, Receptor Logics);human serum albumin scFv fusion (Merrimack); COMBODIES (Epigen Biotech);and IgG/non-IgG fusions (e.g., immunocytokines (EMDSerono, Philogen,ImmunGene, ImmunoMedics).

The terms “treat” and “treatment” refer to the amelioration of one ormore symptoms associated with a cell proliferative disorder; preventionor delay of the onset of one or more symptoms of a cell proliferativedisorder; and/or lessening of the severity or frequency of one or moresymptoms of cell proliferative disorder.

The phrases “to a patient in need thereof”, “to a patient in need oftreatment” or “a subject in need of treatment” includes subjects, suchas mammalian subjects, that would benefit from administration of thecheckpoint regulator antagonist of the present disclosure for treatmentof a cell proliferative disorder.

The terms “therapeutically effective amount”, “pharmacologicallyeffective amount”, and “physiologically effective amount” are usedinterchangeably to mean the amount of a checkpoint regulator antagonistthat is needed to provide a threshold level of active antagonist agentsin the bloodstream or in the target tissue. The precise amount willdepend upon numerous factors, e.g., the particular active agent, thecomponents and physical characteristics of the composition, intendedpatient population, patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein or otherwise available in the relevantliterature.

The terms, “improve”, “increase” or “reduce”, as used in this context,indicate values or parameters relative to a baseline measurement, suchas a measurement in the same individual prior to initiation of thetreatment described herein, or a measurement in a control individual (ormultiple control individuals) in the absence of the treatment describedherein.

A “control individual” is an individual afflicted with the same cellproliferative disorder as the individual being treated, who is about thesame age as the individual being treated (to ensure that the stages ofthe disease in the treated individual and the control individual(s) arecomparable). The individual (also referred to as “patient” or “subject”)being treated may be a fetus, infant, child, adolescent, or adult humanwith a cell proliferative disorder.

The term “cell proliferative disorder” refers to a disordercharacterized by abnormal proliferation of cells. A proliferativedisorder does not imply any limitation with respect to the rate of cellgrowth, but merely indicates loss of normal controls that affect growthand cell division. Thus, in some embodiments, cells of a proliferativedisorder can have the same cell division rates as normal cells but donot respond to signals that limit such growth. Within the ambit of “cellproliferative disorder” is a neoplasm, cancer or tumor.

The term “cancer” refers to any one of a variety of malignant neoplasmscharacterized by the proliferation of cells that have the capability toinvade surrounding tissue and/or metastasize to new colonization sites,and includes leukemia, lymphoma, carcinoma, melanoma, sarcoma, germ celltumor and blastoma. Exemplary cancers for treatment with the methods ofthe instant disclosure include cancer of the brain, bladder, breast,cervix, colon, head and neck, kidney, lung, non-small cell lung,mesothelioma, ovary, prostate, stomach and uterus, leukemia, andmedulloblastoma.

The term “leukemia” refers to progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Exemplary leukemias include, for example, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “carcinoma” refers to the malignant growth of epithelial cellstending to infiltrate the surrounding tissues and give rise tometastases. Exemplary carcinomas include, for example, acinar carcinoma,acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma,alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare,basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolarcarcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriformcarcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloidcarcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma,carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma,cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonalcarcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinomaepitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giantcell carcinoma, carcinoma gigantocellulare, glandular carcinoma,granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

The term “sarcoma” refers to a tumor made up of a substance like theembryonic connective tissue and is generally composed of closely packedcells embedded in a fibrillar or homogeneous substance. Exemplarysarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphomas (e.g., Non-Hodgkin Lymphoma), immunoblastic sarcoma ofT-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parostealsarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma,synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” refers to a tumor arising from the melanocyticsystem of the skin and other organs. Melanomas include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's Disease, multiplemyeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid,premalignant skin lesions, testicular cancer, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, and adrenal corticalcancer.

Checkpoint Regulator Antagonists

The present application provides checkpoint regulator antagonists thatbind specifically to TIGIT, PD-1 and PD-L1 and inhibit one or more oftheir corresponding biological functions. In some embodiments, thecheckpoint regulator antagonist is an anti-TIGIT antibody or antibodyfragment. In some embodiments, the checkpoint regulator antagonist is ananti-PD-1 antibody or antibody fragment. In other embodiments, thecheckpoint regulator antagonist is an anti-PD-L1 antibody or antibodyfragment. In other embodiments, a bispecific or trispecific checkpointregulator antagonist contains multiple specificities (anti-TIGIT,anti-PD-1 and/or anti-PD-L1) for binding several checkpoint regulators.

Anti-TIGIT Antibody and Anti-TIGIT Antibody Fragments

In some embodiments, the TIGIT inhibitor of the present application isan antibody, or an antigen-binding portion thereof, comprising: (1) aheavy chain variable region, wherein the heavy chain variable regioncomprises three complementarity determining regions (HCDRs): HCDR1,HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about80% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 1, 6, 11, 15, 17, 20 and 23, whereinHCDR2 has an amino acid sequence that is about 80% to about 100%homologous to an amino acid sequence selected from the group consistingof SEQ ID NOS: 2, 4, 7, 9, 12, 13, 16, 18, 21 and 24, and wherein HCDR3has an amino acid sequence that is about 80% to about 100% homologous toan amino acid sequence selected from the group consisting of SEQ ID NOS:3, 5, 8, 10, 14, 19, 22 and 25; and (2) a light chain variable region,wherein the light chain variable region comprises three complementaritydetermining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 hasan amino acid sequence that is about 80% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS:26, 29, 31, 33, 35, 39, 42 and 45, wherein LCDR2 has an amino acidsequence that is about 80% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 27, 30, 36,37, 40, 43 and 46, and wherein LCDR3 has an amino acid sequence that isabout 80% to about 100% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 28, 32, 34, 38, 41, 44 and 47,wherein the antibody, or the antigen-binding portion thereof, bindsspecifically to human TIGIT.

In some embodiments, the TIGIT inhibitor of the present application isan antibody, or an antigen-binding portion thereof, comprising (1) aheavy chain variable region having an amino acid sequence that is about80% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 107, 109, 111, 113, 115, 117, 119, 121,123 and 125; and (2) a light chain variable region having an amino acidsequence that is about 80% to about 100% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOS: 108, 110,112, 114, 116, 118, 120, 122, 124 and 126, wherein the antibody, or theantigen-binding portion thereof, binds specifically to human TIGIT.

Anti-PD-1 Antibody and Anti-PD-1 Antibody Fragments

In some embodiments, the PD-1 inhibitor of the present application is anantibody, or an antigen-binding portion thereof, comprising: (1) a heavychain variable region, wherein the heavy chain variable region comprisesthree complementarity determining regions (HCDRs): HCDR1, HCDR2 andHCDR3, wherein HCDR1 has an amino acid sequence that is about 80% toabout 100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 48, 51, 54, 56 and 59, wherein HCDR2 has anamino acid sequence that is about 80% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS:49, 52, 57 and 60, and wherein HCDR3 has an amino acid sequence that isabout 80% to about 100% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 50, 53, 55, 58 and 61; and (2)a light chain variable region, wherein the light chain variable regioncomprises three complementarity determining regions (LCDRs): LCDR1,LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that is about80% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 62, 65, 68, 69, 70 and 73, wherein LCDR2has an amino acid sequence that is about 80% to about 100% homologous toan amino acid sequence selected from the group consisting of SEQ ID NOS:63, 66, 71 and 74, and wherein LCDR3 has an amino acid sequence that isabout 80% to about 100% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 64, 67, 72 and 75, wherein theantibody, or the antigen-binding portion thereof, binds specifically tohuman PD-1.

In some embodiments, the PD-1 inhibitor of the present application is anantibody, or an antigen-binding portion thereof, comprising: (1) a heavychain variable region having an amino acid sequence that is about 80% toabout 100% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 127, 129, 131, 133, 135 and 137; and (2) alight chain variable region having an amino acid sequence that is about80% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 128, 130, 132, 134, 136 and 138, whereinthe antibody, or the antigen-binding portion thereof, binds specificallyto human PD-1.

Anti-PD-L1 Antibody and Anti-PD-L1 Antibody Fragments

In some embodiments, the PD-L1 inhibitor of the present application isan antibody, or an antigen-binding portion thereof, comprising: (1) aheavy chain variable region, wherein the heavy chain variable regioncomprises three complementarity determining regions (HCDRs): HCDR1,HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about80% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 76, 79, 85 and 88, wherein HCDR2 has anamino acid sequence that is about 80% to about 100% homologous to anamino acid sequence selected from the group consisting of SEQ ID NOS:77, 80, 82, 84, 86 and 89, and wherein HCDR3 has an amino acid sequencethat is about 80% to about 100% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOS: 78, 81, 83, 87 and 90;and (2) a light chain variable region, wherein the light chain variableregion comprises three complementarity determining regions (LCDRs):LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that isabout 80% to about 100% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 91, 94, 98, 101 and 104,wherein LCDR2 has an amino acid sequence that is about 80% to about 100%homologous to an amino acid sequence selected from the group consistingof SEQ ID NOS: 92, 95, 99, 102 and 105, and wherein LCDR3 has an aminoacid sequence that is about 80% to about 100% homologous to an aminoacid sequence selected from the group consisting of SEQ ID NOS: 93, 96,97, 100 and 106, wherein the antibody, or the antigen-binding portionthereof, binds specifically to human PD-L1.

In some embodiments, the PD-L1 inhibitor of the present application isan antibody, or an antigen-binding portion thereof, comprising: (1) aheavy chain variable region having an amino acid sequence that is about80% to about 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 139, 141, 143, 145, 147, 149, 151 and153; and (2) a light chain variable region having an amino acid sequencethat is about 80% to about 100% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOS: 140, 142, 144, 146,148, 150, 152 and 154, wherein the antibody, or the antigen-bindingportion thereof, binds specifically to human PD-L1.

The anti-TIGIT, anti-PD-1 and/or anti-PD-L1 antibody can be a monoclonalantibody, chimeric antibody, humanized antibody, scFv or multi-specificantibody comprising additional binding specificities targeting otherTIGIT, PD-1 and/or PD-L1 epitopes or other binding targets as furtherdescribed below. FIG. 4 shows the amino acid sequences of the CDRscorresponding to anti-TIGIT, anti-PD-1 and anti-PD-L1 monoclonalantibodies according to the present application.

The HCVRs and LCVRs described herein may be linked to anaturally-occurring Fc region or a non-naturally occurring or mutated Fcregion, e.g., an effectorless or mostly effectorless Fc (e.g., humanIgG2 or IgG4) or, alternatively, an Fc with enhanced binding to one ormore activating Fc receptors (FcγRI, FcγRIIa or FcγRIIIa) so as toenhance T_(reg) depletion in the tumor environment.

Accordingly, in certain embodiments the anti-TIGIT, anti-PD-1 and/oranti-PD-L1 HCVRs and LCVRs described herein may be linked to an Fccomprising one or more modifications, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody described herein may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or it may be modified to alter its glycosylation, toalter one or more functional properties of the antibody. Morespecifically, in certain embodiments, the antibodies in the presentapplication may include modifications in the Fc region in order togenerate an Fc variant with (a) increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) increased ordecreased affinity for C1q and/or (d) increased or decreased affinityfor a Fc receptor relative to the parent Fc. Such Fc region variantswill generally comprise at least one amino acid modification in the Fcregion. Combining amino acid modifications is thought to be particularlydesirable. For example, the variant Fc region may include two, three,four, five, etc. substitutions therein, e.g., of the specific Fc regionpositions identified herein.

For uses where effector function is to be avoided altogether, e.g., whenantigen binding alone is sufficient to generate the desired therapeuticbenefit, and effector function only leads to (or increases the risk of)undesired side effects, IgG4 antibodies may be used, or antibodies orfragments lacking the Fc region or a substantial portion thereof can bedevised, or the Fc may be mutated to eliminate glycosylation altogether(e.g., N297A). Alternatively, a hybrid construct of human IgG2 (CH1domain and hinge region) and human IgG4 (CH2 and CH3 domains) may begenerated that is devoid of effector function, lacking the ability tobind FcγRs (like IgG2) and activate complement (like IgG4). When usingan IgG4 constant domain, it is usually preferable to include thesubstitution S228P, which mimics the hinge sequence in IgG₁ and therebystabilizes IgG4 molecules, reducing Fab-arm exchange between thetherapeutic antibody and endogenous IgG4 in the patient being treated.

In certain embodiments, the anti-TIGIT, anti-PD-1 or anti-PD-L1 antibodyor fragment thereof may be modified to increase its biologicalhalf-life. Various approaches may be employed, including e.g., thatincrease the binding affinity of the Fc region for FcRn. In oneembodiment, the antibody is altered within the CH1 or CL region tocontain a salvage receptor binding epitope taken from two loops of a CH2domain of an Fc region of an IgG, as described in U.S. Pat. Nos.5,869,046 and 6,121,022. The numbering of residues in the Fc region isthat of the EU index of Kabat. Sequence variants disclosed herein areprovided with reference to the residue number followed by the amino acidthat is substituted in place of the naturally occurring amino acid,optionally preceded by the naturally occurring residue at that position.Where multiple amino acids may be present at a given position, e.g., ifsequences differ between naturally occurring isotypes, or if multiplemutations may be substituted at the position, they are separated byslashes (e.g., “X/Y/Z”).

Exemplary Fc variants that increase binding to FcRn and/or improvepharmacokinetic properties include substitutions at positions 259, 308,and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F,434Y, and 434M. Other variants that increase Fc binding to FcRn include:250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem.279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology176:346-356), 256A, 272A, 305A, 307A, 31 IA, 312A, 378Q, 380A, 382A,434A (Shields et al. (2001) J. Biol. Chem., 276(9):6591-6604), 252F,252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E,433K/434F/436H (Dall'Acqua et al. (2002) J. Immunol., 169:5171-5180,Dall'Acqua et al. (2006) J. Biol. Chem., 281:23514-23524, and U.S. Pat.No. 8,367,805.

Modification of certain conserved residues in IgG Fc (1253, H310, Q311,H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol.182:7663), have been proposed as a way to increase FcRn affinity, thusincreasing the half-life of the antibody in circulation (WO 98/023289).The combination Fc variant comprising M428L and N434S has been shown toincrease FcRn binding and increase serum half-life up to five-fold(Zalevsky et al. (2010) Nat. Biotechnol. 28:157). The combination Fcvariant comprising T307A, E380A and N434A modifications also extendshalf-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol.18:1759). In addition, combination Fc variants comprising M252Y-M428L,M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, andM428L-N434S variants have also been shown to extend half-life (U.S.2006/173170). Further, a combination Fc variant comprising M252Y, S254Tand T256E was reported to increase half-life-nearly 4-fold. Dall'Acquaet al. (2006) J. Biol. Chem. 281:23514.

Bispecific Checkpoint Regulator Antagonists

Another aspect of the present application relates to a bispecificcheckpoint regulator antagonist, comprising: a first targeting domainthat binds specifically to a checkpoint regulator and a second targetingdomain that binds specifically to a second target. In some embodiments,the second target is selected from the group consisting of vascularendothelial growth factor (VEGF), vascular endothelial growth factorreceptor (VEGFR), immune checkpoint regulators, Tie2 tyrosine kinasereceptors and ligands of Tie2 tyrosine kinase receptors.

In some embodiments, the second target is also a checkpoint regulator.

In some embodiments, the first targeting domain comprises theantigen-binding portion of the anti-TIGIT, anti-PD-1 and/or anti-PD-L1antibody of the present application. In some embodiments, the secondtargeting domain comprises the antigen-binding portion of anotheranti-TIGIT, anti-PD-1 antibody and/or anti-PD-L1 antibody of the presentapplication. In some embodiments, the first targeting domain and thesecond targeting domain each comprises the antigen-binding portion of ananti-TIGIT, anti-PD-1 and/or anti-PD-L1 antibody of the presentapplication, wherein the first targeting domain is different from thesecond targeting domain.

In some embodiments, the bispecific checkpoint regulator antagonistcomprises a first targeting domain that binds specifically to humanTIGIT and a second targeting domain that binds specifically to PD-1 orPD-L1.

In some embodiments, the bispecific checkpoint regulator antagonistcomprises a one or more PD-1-specific CDRs of FIG. 3B or may include oneor more PD-L1-specific CDRs of FIG. 3C. In some embodiments, thebispecific checkpoint regulator antagonist comprises a one or morePD-1-specific variable regions of FIGS. 4D-E or one or more PD-L1specific variable regions of FIG. 4F-H.

In some embodiments, the bispecific checkpoint regulator antagonist is afull-length antibody that binds human TIGIT on one of its two bindingarms (one pair of HC/LC), and binds a different antigen (or epitope) onits second arm (a different pair of HC/LC). In these embodiments, thebispecific antibody has two distinct antigen binding arms (in bothspecificity and CDR sequences), and is monovalent for each antigen itbinds to.

In other embodiments, the bispecific checkpoint regulator antagonist isa full-length antibody that can bind human TIGIT and other antigens ineach of its two binding arms (a pair of HC/LC). In these embodiments,the bispecific checkpoint regulator antagonist has two identical antigenbinding arms, with identical specificity and identical CDR sequences,and is bivalent for each antigen it binds to.

FIGS. 17A-17N show a variety of different bispecific checkpointregulator antagonists, M1-M14, where: (1) the VH1 and VL1 regionscorrespond to anti-PD1, anti-PD-L1 or any other mab variable domain; and(2) the VH2 and VL2 regions correspond to anti-TIGIT variable domains.FIGS. 17A-17D depict exemplary bispecific checkpoint regulatorantagonist embodiments comprising a homodimer scaffold containing doublechain arms with dual binding specificities (anti-TIGIT and anti-PD-1).FIGS. 17E and 17F depict exemplary bispecific checkpoint regulatorantagonist embodiments comprising a homodimer scaffold containing a pairof double chain arms with a first binding specificity at the N-terminalof the constant regions and a pair of double chain arms with a secondbinding specificity at the C-terminal of the constant regions. FIGS.17G-17J depict exemplary bispecific checkpoint regulator antagonistembodiments comprising a homodimer scaffold containing a pair of doublechain arms with a first binding specificity at the N-terminal of theconstant regions and a pair of single chain Fv (ScFv) fragments with asecond binding specificity) at the C-terminal of the constant regions.FIGS. 17K-17N depict exemplary bispecific checkpoint regulatorantagonist embodiments comprising a heterodimer scaffold containing apair of double chain arms each having a different binding specificity.In these figures, the stars designate mutations in immunoglobulinconstant regions that prevent mispairing of light or heavy chains fromone binding specificity with light or heavy chains from another bindingspecificity as further discussed below.

Exemplary bispecific checkpoint regulator antagonist sequences are shownin FIG. 5 . In one embodiment, a bispecific checkpoint regulatorantagonist, TP-M2T8P5 comprises: (1) a heavy chain variable regionhaving an amino acid sequence that is about 80% to about 100% homologousto the amino acid sequence in SEQ ID NO: 155; and (2) a variable lightchain variable region having an amino acid sequence that is about 80% toabout 100% homologous to the amino acid sequence in SEQ ID NO: 156.

In another embodiment, a bispecific checkpoint regulator antagonist,TP-M4T8P5 comprises: (1) a light chain variable region 2/heavy chainvariable region 1 (VL2/VH1) having an amino acid sequence that is about80% to about 100% homologous to the amino acid sequence in SEQ ID NO:157; and (2) a heavy chain variable 2 region/light chain variable region1 (VH2/VL1) having an amino acid sequence that is about 80% to about100% homologous to the amino acid sequence in SEQ ID NO: 158.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M6T8P5 comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80% to about 100% homologous to the aminoacid sequence in SEQ ID NO: 159; (2) a light chain variable region2/light chain constant region (VL2/CL) having an amino acid sequencethat is about 80% to about 100% homologous to the amino acid sequence inSEQ ID NO: 160; and (3) a heavy chain variable region 1/heavy chainconstant region 1 (VH1/CH1) having an amino acid sequence that is about80% to about 100% homologous to the amino acid sequence in SEQ ID NO:161.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M6T8L8 comprises: (1) a heavy chain variable region having an aminoacid sequence that is about 80% to about 100% homologous to the aminoacid sequence in SEQ ID NO: 162; (2) a light chain variable region2/light chain constant region (VL2/CL) having an amino acid sequencethat is about 80% to about 100% homologous to the amino acid sequence inSEQ ID NO: 163; and (3) a heavy chain variable region 1/heavy chainconstant region 1 (VH1/CH1) having an amino acid sequence that is about80% to about 100% homologous to the amino acid sequence in SEQ ID NO:164.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M8T10P1 (TP-83) comprises: (1) a heavy chain variable region havingan amino acid sequence that is about 80% to about 100% homologous to theamino acid sequence in SEQ ID NO: 165; and (2) a light chain having anamino acid sequence that is about 80% to about 100% homologous to theamino acid sequence in SEQ ID NO: 166.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M9T10P1 (TP-93) comprises: (1) a heavy chain that is about 80% toabout 100% homologous to the amino acid sequence in SEQ ID NO: 167; and(2) a light chain having an amino acid sequence that is about 80% toabout 100% homologous to the amino acid sequence in SEQ ID NO: 168.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M9T10P5 comprises: (1) a heavy chain that is about 80% to about 100%homologous to the amino acid sequence in SEQ ID NO: 169; and (2) a lightchain having an amino acid sequence that is about 80% to about 100%homologous to the amino acid sequence in SEQ ID NO: 170.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M10T8P5 (TP-92) comprises: (1) a heavy chain that is about 80% toabout 100% homologous to the amino acid sequence in SEQ ID NO: 171; and(2) a light chain variable region having an amino acid sequence that isabout 80% to about 100% homologous to the amino acid sequence in SEQ IDNO: 172.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M10T8L8 comprises: an amino acid sequence that is about 80% to about100% homologous to the amino acid sequence in SEQ ID NO: 173.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M14T8P5 comprises: (1) a heavy chain variable region 2/heavy chainconstant region 1 (VH2/CH1) having an amino acid sequence that is about80% to about 100% homologous to the amino acid sequence in SEQ ID NO:174; (2) a variable light chain region 1/heavy chain constant region 1(VL1/CH1) having an amino acid sequence that is about 80% to about 100%homologous to the amino acid sequence in SEQ ID NO: 175; (3) a lightchain variable region 2/light chain constant region (VL2/CL) having anamino acid sequence that is about 80% to about 100% homologous to theamino acid sequence in SEQ ID NO: 176; and (4) a heavy chain variableregion 1/light chain constant region (VH1/CL) having an amino acidsequence that is about 80% to about 100% homologous to the amino acidsequence in SEQ ID NO: 177.

In another embodiment, the bispecific checkpoint regulator inhibitor,TP-M14T8L8 comprises: (1) a heavy chain variable region 2/heavy chainconstant region 1 (VH2/HC1) having an amino acid sequence that is about80% to about 100% homologous to the amino acid sequence in SEQ ID NO:178; (2) a variable light chain region 1/heavy chain constant region 2(VL1/HC2) having an amino acid sequence that is about 80% to about 100%homologous to the amino acid sequence in SEQ ID NO: 179; (3) a lightchain variable region 2/light chain constant region (VL2/CL) having anamino acid sequence that is about 80% to about 100% homologous to theamino acid sequence in SEQ ID NO: 180; and (4) a heavy chain variableregion 1/light chain constant region (VH1/CL) having an amino acidsequence that is about 80% to about 100% homologous to the amino acidsequence in SEQ ID NO: 181.

Trispecific Checkpoint Regulator Antagonists

In some embodiments, the trispecific checkpoint regulator antagonistcomprises: (1) a first targeting domain having a first bindingspecificity conferred by one or more anti-TIGIT variable domains; (2) asecond targeting conferred by one or more variable regions comprisingone or more anti-PD1 variable domains, one or more anti-PD-L1 variabledomains, or one or more variable region domains corresponding to anyother anti-cancer targets, including other checkpoint regulators, asfurther described herein; and (3) a third targeting domain conferring byone or more peptide sequences (e.g., AMG386) or variable domain regionscomprising a VEGF binding antagonist, Tie2 tyrosine kinase receptorantagonist or Tie2 tyrosine kinase receptor ligand antagonist as furtherdescribed herein.

FIGS. 18A-18J show a variety of different trispecific checkpointregulator antagonists, where: (1) the VH1 and VL1 regions correspond toanti-TIGIT variable domains; (2) the VH2 and VL21 regions correspond toanti-PD1, anti-PD-L1 or any other mab variable domain; and (3) thecircular region corresponds to AMG386 or any other biological peptide.

Homedimers and Hetrodimers

One of the challenges for efficiently producing bispecific andtrispecific checkpoint regulator antagonist preparations concernsmispairing of heavy and light chains when co-expressing chains ofdifferent binding specificities. Table 1 lists several amino acidsubstitution options for overcoming mispairing between heavy chains ofdifferent binding specificities, which “enforce” or preferentiallypromote correct association between desired heavy chains. Any approachto prevent or reduce mispairing between heavy chains may be used to makethe bispecific or trispecific checkpoint regulator antagonists accordingto the present disclosure.

The “knobs-into-hole” (KiH) approach relies on modifications of theinterface between the two CH3 domains where most interactions occur.Typically, a bulky residue is introduced into the CH3 domain of oneantibody heavy chain and acts similarly to a key. In the other heavychain, a “hole” is formed that is able to accommodate this bulkyresidue, mimicking a lock. The resulting heterodimeric Fc-part can befurther stabilized by artificial disulfide bridges.

An alternative approach is based on charged residues with ionicinteractions or steric complementarity. This includes altering thecharge polarity in the CH3 interface so that co-expression ofelectrostatically matched Fc domains support favorable attractiveinteractions and heterodimer formation while retaining the hydrophobiccore, whereas unfavorable repulsive charge interactions suppresshomodimerization. See Table 1. The amino acid numbering in Table 1follows the Kabat numbering scheme and can be applied to heavy chainamino acid sequences of the antibodies described herein.

In a further approach, leucine zipper (LZ) domains may be incorporatedinto a protein scaffold. A leucine zipper is a common three-dimensionalstructural motif in proteins, typically as part of a DNA-binding domainin various transcription factors. A single LZ typically contains 4-5leucine residues at approximately 7-residue intervals, which forms anamphipathic alpha helix with a hydrophobic region running along oneside. In a particular embodiment, a heterodimeric protein scaffoldcomprises a LZ from the c-jun transcription factor associated with a LZfrom the c-fos transcription factor. Although c-jun is known to formjun-jun homodimers and c-fos does not form homodimers, the formation ofjun-fos heterodimers is greatly favored over jun-jun homodimers.

A leucine zipper domain may be incorporated in place of CH2-CH3sequences in the protein scaffold or it may be placed at the carboxyterminal end of the two heavy chains in the bispecific or trispecificcheckpoint regulator antagonist. In the case of the latter, a furincleavage site may be introduced between the carboxy terminal end of CH3and the amino terminal end of the leucine zipper. This can facilitatefurin-mediated cleavage of the leucine zipper following theheterodimerization step when co-expressing the heavy and light chains ofthe bispecific or trispecific checkpoint regulator antagonist in anappropriate mammalian cell expression system (see Wranik et al., J.Biol. Chem., 287(5):43331-43339, 2012).

TABLE 1 Type HC1 HC2 Knobs-into-holes Y349C, T366S, S354C, T366W L368A,Y407V Ionic, electrostatic S183E, E356K, S183K, K370E, E357K, D399KK409D, K439E Ionic, electrostatic K392D, K404D E356K, D399K HA-TFsubstitutions S364H, F405A Y349T, T394F HF-TA substitutions S364H, T394FY349T, F405A Leucine zipper human c-Jun human c-fos heterodimer leucinezipper leucine zipper

The amino acid numbering in Table 1 follows the Kabat numbering schemeand can be applied to heavy chain amino acid sequences of the antibodiesdescribed herein. The mutations described in Table 1 may be applied tothe sequence (published or otherwise) of any immunoglobulin IgG1 heavychain, as well as other immunoglobulin classes, and subclasses (orisotypes) therein.

When co-expressing heavy and light chains of bispecific or trispecificantibodies, the light chains of one binding specificity can also mispairwith heavy chains of a different binding specificity. Therefore, incertain embodiments, portions of the heavy chain, light chain or bothmay be modified relative to the “wild-type” antibody chains from whichthey are derived to prevent or reduce mispairing of both heavy chainconstant regions to one another, as well mispairing of light chainconstant regions to their heavy chain counterparts.

The light chain mispairing problem can be addressed in several ways. Insome embodiments, sterically complementary mutations and/or disulfidebridges may be incorporated into the two VL/VH interfaces. In otherembodiments, mutations can be incorporated based on ionic orelectrostatic interactions. In some embodiments, light chain mispairingmay be prevented or reduced by employing a first arm with an S183Emutation in the CH1 domain of the heavy chain and an S176K mutation inthe CL domain of the light chain. A second arm may include an S183Kmutation in the in the CH1 domain of the heavy chain and an S176Emutation in the CL domain of the light chain. In other embodiments, a“CrossMab” approach is employed, where one arm in the bispecific ortrispecific checkpoint regulator antagonist (e.g., Fab) is leftuntouched, but in the other arm containing the other bindingspecificity, one or more domains in the light chain are swapped with oneor more domains in the heavy chain at the heavy chain: light chaininterface.

Methods, immunoglobulin domain sequences, including specific mutationsfor preventing mispairing of heavy and light chains as disclosed aboveare further described in U.S.

Patent Application Publication Nos. 2014/0243505, 2013/0022601.

FIGS. 18G-18J depict exemplary trispecific checkpoint regulatorantagonist embodiments in which the stars designate mutations inimmunoglobulin constant regions that prevent mispairing of light orheavy chains from one binding specificity with light or heavy chainsfrom another binding specificity.

Conjugates

In certain embodiments, the checkpoint regulator antagonists of thepresent application are chemically conjugated to one or more peptidesand/or small molecule drugs. The peptides or small molecule drug can bethe same or different. The peptides or small molecule drugs can beattached, for example to reduced SH groups and/or to carbohydrate sidechains. Methods for making covalent or non-covalent conjugates ofpeptides or small molecule drugs with antibodies are known in the artand any such known method may be utilized.

In some embodiments the peptide or small molecule drug is attached tothe hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached using aheterobifunctional cross-linkers, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). General techniques for suchconjugation are well-known in the art. In some embodiments, the peptideor small molecule drug is conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same agent that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different therapeutic ordiagnostic agent. Methods for conjugating peptide inhibitors or smallmolecule drugs to antibodies via antibody carbohydrate moieties iswell-known to those of skill in the art. For example, in one embodiment,the method involves reacting an antibody component having an oxidizedcarbohydrate portion with a carrier polymer that has at least one freeamine function. This reaction results in an initial Schiff base (imine)linkage, which can be stabilized by reduction to a secondary amine toform the final conjugate. Exemplary methods for conjugating smallmolecule drugs and peptides to antibodies are described in U.S. PatentApplication Publication No. 2014/0356385.

Preferably, the checkpoint regulator antagonists in the presentdisclosure retain certain desirable characteristics and pharmacokineticproperties of antibodies, including a desirable in vitro and in vivostability (e.g., lone half-life and shelf-life stability), efficientdelivery into desired target cells, increased affinity for bindingpartners, desirable antibody-dependent cell-mediated cytotoxicity andcomplement-dependent cytotoxicity, and reduced renal clearance orexcretion. Accordingly, careful attention to size and need forparticular constant region effector functions may be considered in thedesign of the checkpoint regulator antagonists.

The anti-TIGIT, anti-PD-1 and anti-PD-L1 inhibitors, includingmonospecific, bispecific and trispecific checkpoint regulatorantagonists therefrom, may range in size from 50 kD to 300 kD, from 50kD to 250 kD, from 60 kD to 250 kD, from 80 kDa to 250 kD, from 100 kDto 250 kD, from 125 kD to 250 kD, from 150 kD to 250 kD, from 60 kD to225 kD, from 75 kD to 225 kD, from 100 kD to 225 kD, from 125 kD to 225kD, from 150 kD to 225 kD, from 60 kD to 200 kD, from 75 kD to 200 kD,from 100 kD to 125 kD to 200 kD, from 150 kD to 200 kD, from 60 kD to150 kD, from 75 kD to 150 kD, from 100 kD to 150 kD, from 60 kD to 125kD, from 75 kD to 125 kD, from 75 kD to 100 kD, or any range encompassedby any combination of whole numbers listed in the above cited ranges orany ranges specified by any combination of whole numbers between any ofthe above cited ranges.

Kits

The present application further provides a kit comprising the checkpointregulator antagonist of the present application. In some embodiment, thekit comprises one or more of the anti-TIGIT, anti-PD-1 or anti-PD-L1antibodies, and bispecific or trispecific checkpoint regulatorantagonists thereof. In some embodiments, the kit further containsadditional reagents, including secondary antibodies for detection, andadditional human antibodies described herein (e.g., a human antibodyhaving a complementary activity that binds to an epitope in TIGITantigen distinct from the first human antibody). Kits typically includea label with instructions indicating the intended use of the contents ofthe kit. The term label includes any writing, or recorded materialsupplied on or with the kit, or which otherwise accompanies the kit.

Methods of Using the Checkpoint Regulator Antagonists

The checkpoint regulator antagonists of the present application, such asanti-TIGIT antibodies, anti-TIGIT antibody fragments, anti-PD-1antagonists, anti-PD-1 antibody fragments, anti-PD-L1 antibodies,anti-PD-L1 antibody fragments, bispecific checkpoint regulatorantagonists and trispecific checkpoint regulator antagonists that bindspecifically to TIGIT, PD-1 and/or PD-L1, have numerous in vitro and invivo utilities including, for example, enhancement of immune responsesby blocking signaling by TIGIT, PD-1 and/or PD-L1, treatment of cancers,infectious diseases or autoimmune diseases, and detection of TIGIT, PD-1and/or PD-L1.

In some embodiments, the checkpoint regulator antagonists of the presentapplication are administered to cells in culture, in vitro or ex vivo,or to human subjects, e.g., in vivo, to enhance immunity in a variety ofdiseases. Accordingly, provided herein are methods of modifying animmune response in a subject comprising administering to the subject anantibody or antigen-binding fragment thereof as described herein suchthat the immune response in the subject is enhanced, stimulated orup-regulated. Preferred subjects include human patients in whomenhancement of an immune response would be desirable. The methods areparticularly suitable for treating human patients having a disorder thatcan be treated by augmenting an immune response (e.g., the T-cellmediated immune response). The methods are particularly suitable fortreatment of cancer or chronic infections in vivo. For example, theanti-TIGIT, anti-PD-1 or anti-PD-L1 compositions may be administeredtogether with an antigen of interest or the antigen may already bepresent in the subject to be treated (e.g., a tumor-bearing orvirus-bearing subject) to enhance antigen-specific immunity. Whenanti-TIGIT antibodies are administered together with another agent, thetwo can be administered separately or simultaneously.

In some embodiments, the checkpoint regulator anatagonist used in theabove-described method is an anti-TIGIT, anti-PD-1, anti-PD-L1 antibody,fragment thereof, or combination thereof. In some embodiments, thecheckpoint regulator anatagonist is a bispecific or trispecific antibodyof the present application.

In some embodiments, the checkpoint regulator antagonist is an antibodyor fragment. In some embodiments, the antibodies described herein arehuman or humanized antibodies.

Also encompassed are methods for detecting and/or measuring the presenceof human TIGIT, human PD-1 or human PD-L1 in a sample comprisingcontacting the sample, and a control sample, with a human monoclonalantibody thereof, or an antigen binding fragment thereof, whichspecifically binds to human TIGIT, human PD-1 or human PD-L1 underconditions that allow for formation of a complex between the antibody orfragment thereof and human TIGIT, human PD-1 or human PD-L1. Theformation of a complex is then detected, wherein a difference in complexformation between the sample compared to the control sample isindicative the presence of human TIGIT antigen in the sample. Moreover,the anti-TIGIT, anti-PD-1 or anti-PD-L1 antibody described herein can beused to purify human TIGIT via immunoaffinity purification.

Given the ability of anti-TIGIT, anti-PD-1 and anti-PD-L1 antibodies toblock inhibition or co-inhibition of T cell responses, e.g.,antigen-specific T cell responses, provided herein are in vitro and invivo methods of using the antibodies described herein to stimulate,enhance or upregulate antigen-specific T cell responses, e.g.,anti-tumor T cell responses. In certain embodiments, CD3 stimulation isalso provided (e.g., by co-incubation with a cell expressing membraneCD3), which stimulation can be provided at the same time, before, orafter treatment with an anti-TIGIT, anti-PD-1 or anti-PD-L1 antibody.For example, provided herein are methods of enhancing anantigen-specific T cell response comprising contacting said T cell withan anti-TIGIT, anti-PD-1 or anti-PD-L1 antibody described herein, andoptionally with CD3, such that an antigen-specific T cell response isenhanced, e.g., by removal of a TIGIT-, PD-1 or PD-L1 mediatedinhibitory effect. Any suitable indicator of an antigen-specific T cellresponse can be used to measure the antigen-specific T cell response.Non-limiting examples of such suitable indicators include increased Tcell proliferation in the presence of the antibody and/or increasecytokine production in the presence of the antibody. In a preferredembodiment, interleukin-2 and/or interferon-7 production by theantigen-specific T cell is enhanced.

Further encompassed are methods for enhancing an immune response (e.g.,an antigen-specific T cell response) in a subject comprisingadministering an anti-TIGIT antibody, an anti-PD-1 antibody, ananti-PD-L1 antibody, or a bispecific or trispecific checkpoint regulatorantagonist described herein to the subject such that an immune response(e.g., an antigen-specific T cell response) in the subject is enhanced.In a preferred embodiment, the subject is a tumor-bearing subject and animmune response against the tumor is enhanced. A tumor may be a solidtumor or a liquid tumor, e.g., a hematological malignancy. In certainembodiments, a tumor is an immunogenic tumor. In other embodiments, atumor is non-immunogenic. In certain embodiments, a tumor is PD-L1positive. In other embodiments a tumor is PD-L1 negative. A subject mayalso be a virus-bearing subject in which an immune response against thevirus is enhanced as a consequence of administering an anti-TIGITantibody, anti-PD-1 antibody, anti-PD-L1 antibody, bispecific checkpointregulator antagonist, or trispecific checkpoint regulator antagonist asdescribed herein.

In one embodiment, a method for inhibiting the growth of tumor cells ina subject comprises administering to the subject an anti-TIGIT antibody,anti-PD-1 antibody, anti-PD-L1 antibody, bispecific checkpoint regulatorantagonist, or trispecific checkpoint regulator antagonist describedherein such that growth of the tumor is inhibited in the subject. Alsoprovided are methods of treating chronic viral infection in a subjectcomprising administering to the subject an anti-TIGIT antibody,anti-PD-1 antibody, anti-PD-L1 antibody, bispecific checkpoint regulatorantagonist, or trispecific checkpoint regulator antagonist as describedherein such that the chronic viral infection is treated in the subject.

Also encompassed herein are methods for depleting T_(Kg) cells from thetumor microenvironment of a subject with a tumor, e.g., cancerous tumor,comprising administering to the subject a therapeutically effectiveamount of an anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1antibody, bispecific checkpoint regulator antagonist, or trispecificcheckpoint regulator antagonist described herein that comprises an Fcthat stimulates depletion of T_(reg) cells in the tumormicroenvironment. An Fc may, e.g., be an Fc with effector function orenhanced effector function, such as binding or having enhanced bindingto one or more activating Fc receptors.

In a preferred embodiment, T_(reg) depletion occurs without significantdepletion or inhibition of T_(eff) in the tumor microenvironment, andwithout significant depletion or inhibition of T_(eff) cells and T_(reg)cells outside of the tumor microenvironment. In certain embodiments, thesubject has higher levels of TIGIT on T_(reg) cells than on T_(eff)cells, e.g., in the tumor microenvironment. In certain embodiments,anti-TIGIT antibodies or antagonists may deplete T_(regs) in tumorsand/or T_(regs) in tumor infiltrating lymphocytes (TILs). For example,in the CT26 tumor model, an anti-mouse TIGIT antibody formatted as amouse IgG2a (which exhibits effector function) partially depleted bothT_(reg) and CD8⁺ T cells, but did not deplete CD4⁺ T cells. Aneffectorless counterpart anti-TIGIT antibody, formatted as a mouse IgG1D265A, did not deplete T cells.

When considering whether or not to employ Fc effector function or aneffectorless anti-TIGIT antibody, due consideration must be given to thetradeoff between depletion of T_(regs), which may enhance anti-tumorimmune response, and depletion of CD8⁺ T cells, which would eliminatesome of the cells needed to actually kill tumor cells. Althoughdepletion of T_(reg)s might be expected to enhance anti-tumor activity,recent studies have demonstrated that ligation of TIGIT on TIGIT⁺T_(regs) promotes T_(reg) cell-mediated suppression of T_(eff) cellproliferation (Joller et al. (2014) Immunity 40:569), suggesting thatblocking of TIGIT signaling (e.g., using an antagonist anti-TIGITantibody of the present invention) might also enhance anti-tumoractivity. Accordingly, it may be most efficacious to use an antagonistanti-TIGIT antibody lacking effector function, which: i) blocks TIGITsignaling in T_(regs) thus reducing their immunosuppressive activity;ii) activates anti-tumor CD8+ Tcells by blocking TIGIT's inhibitoryeffects, while at the same time avoiding theireffector-function-mediated depletion; and iii) enhances DNAM-mediatedactivation by allowing DNAM to bind to PVR (CD155, the TIGIT ligand)that would otherwise have been bound by TIGIT (and by reducing directTIGIT-DNAM interactions) (Johnston et al. (2014) Cancer Cell 26:923).The same is applicable to use of anti-PD-1 antibodies, anti-PD-L1antibodies, bispecific checkpoint regulator antagonists, or trispecificcheckpoint regulator antagonists.

In certain embodiments, an anti-TIGIT antibody, anti-PD-1 antibody,anti-PD-L1 antibody, bispecific checkpoint regulator antagonist, ortrispecific checkpoint regulator antagonist described herein is given toa subject as an adjunctive therapy. Treatment of cancer patient with ananti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, bispecificcheckpoint regulator antagonist, or trispecific checkpoint regulatorantagonist according to the present application may lead to a long-termdurable response relative to the current standard of care; long termsurvival of at least 1, 2, 3, 4, 5, 10 or more years, recurrence freesurvival of at least 1, 2, 3, 4, 5, or 10 or more years. In certainembodiments, treatment of a cancer patient with an anti-TIGIT antibody,anti-PD-1 antibody, anti-PD-L1 antibody, bispecific checkpoint regulatorantagonist, or trispecific checkpoint regulator antagonist preventsrecurrence of cancer or delays recurrence of cancer by, e.g., 1, 2, 3,4, 5, or 10 or more years. An anti-TIGIT, anti-PD-1 and/or anti-PD-L1treatment can be used as a primary or secondary line of treatment.

In certain preferred embodiments, the subject has a cell proliferativedisease or cancer. Blocking of PVR/Nectin-2 signaling through TIGIT byanti-TIGIT antibodies can enhance the immune response to cancerous cellsin the patient. Similarly, blocking of Provided herein are methods fortreating a subject having cancer, comprising administering to thesubject an anti-TIGIT, anti-PD-1, anti-PD-L1, bispecific checkpointregulator antagonist, or trispecific checkpoint regulator antagonistthereof as described herein, such that the subject is treated, e.g.,such that growth of cancerous tumors is inhibited or reduced and/or thatthe tumors regress. An anti-TIGIT anti-PD-1, anti-PD-L1, bispecificcheckpoint regulator antagonist, or trispecific checkpoint regulatorantagonist thereof as described herein can be used alone to inhibit thegrowth of cancerous tumors. Alternatively, any of these checkpointregulator antagonists can be used in conjunction with another agent,e.g., other anti-cancer targets, immunogenic agents, standard cancertreatments, or other antibodies, as described below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-TIGIT,anti-PD-1, anti-PD-L1, bispecific checkpoint regulator antagonist, ortrispecific checkpoint regulator antagonist as described herein.Preferably, the antibody is a human anti-TIGIT, anti-PD-1 or anti-PD-L1antibody comprising the anti-TIGIT, anti-PD-1 or anti-PD-L1 HCVRs andLCVR described herein, or it may be a chimeric or humanized non-humananti-hu TIGIT, anti-hu PD-1 or anti-PD-L1 antibody, e.g., a chimeric orhumanized anti-TIGIT, anti-PD-1 or anti-PD-L1 antibody that competes forbinding with, or binds to the same epitope as, at least one of theanti-TIGIT, anti-PD-1 or anti-PD-L1 antibodies described herein.

Cancers whose growth may be inhibited using the antibodies of theinvention include cancers typically responsive to immunotherapy.Non-limiting examples of cancers for treatment include squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, squamousnon-small cell lung cancer (NSCLC), non NSCLC, glioma, gastrointestinalcancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer (e.g.,renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractoryprostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, andhead and neck cancer (or carcinoma), gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastaticmalignant melanoma, such as cutaneous or intraocular malignantmelanoma), bone cancer, skin cancer, uterine cancer, cancer of the analregion, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, cancer ofthe ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally-induced cancers including those induced by asbestos,virus-related cancers (e.g., human papilloma virus (HPV)-related tumor),and hematologic malignancies derived from either of the two major bloodcell lineages, i.e., the myeloid cell line (which produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cellline (which produces B, T, NK and plasma cells), such as all types ofleukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocyticand/or myelogenous leukemias, such as acute leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), andchronic myelogenous leukemia (CIVIL), undifferentiated AML (MO),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NEIL), B-celllymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoidB-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma,anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-celllymphoma/leukemia, mantle cell lymphoma, angio immunoblastic T-celllymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primarymediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma,T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-celllymphoma, lymphoblastic lymphoma, post-transplantationlymphoproliferative disorder, true histiocytic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, lymphoblasticlymphoma (LBL), hematopoietic tumors of lymphoid lineage, acutelymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides orSezary syndrome), and lymphoplasmacytoid lymphoma (LPL) withWaldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, lightchain myeloma, nonsecretory myeloma, smoldering myeloma (also calledindolent myeloma), solitary plasmocytoma, and multiple myelomas, chroniclymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, including fibrosarcomaand rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the centraland peripheral nervous, including astrocytoma, schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, hematopoietic tumors of lymphoid lineage, for exampleT-cell and B-cell tumors, including but not limited to T-cell disorderssuch as T-prolymphocytic leukemia (T-PLL), including of the small celland cerebriform cell type; large granular lymphocyte leukemia (LGL)preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head orneck, renal cancer, rectal cancer, cancer of the thyroid gland; acutemyeloid lymphoma, as well as any combinations of said cancers. Themethods described herein may also be used for treatment of metastaticcancers, refractory cancers (e.g., cancers refractory to previousimmunotherapy, e.g., with a blocking CTLA-4 or PD-1 antibody), andrecurrent cancers.

An anti-TIGIT, anti-PD-1 antibody, anti-PD-L1 antibody, bispecificcheckpoint regulator antagonist, or trispecific checkpoint regulatorantagonist can be administered alone, in combination with anothercheckpoint regulator antagonist, or concurrently with another checkpointregulator antagonist. An anti-TIGIT, anti-PD-1 antibody, anti-PD-L1antibody, bispecific checkpoint regulator antagonist, or trispecificcheckpoint regulator antagonist can also be administered in combination,or concurrently with, an immunogenic agent, such as cancerous cells,tumor vaccines, purified tumor antigens (including recombinant proteins,peptides, and carbohydrate molecules), cells transfected with genesencoding immune stimulating cytokines, in a cancer vaccine strategy (Heet al. (2004) J. Immunol. 173:4919-28), or an oncolytic virus.

Many experimental strategies for vaccination against tumors have beendevised. In one of these strategies, a vaccine is prepared usingautologous or allogeneic tumor cells. Some of these cellular vaccineshave been shown to be most effective when the tumor cells are transducedto express GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90: 3539-43). Cancer vaccines have been shown toenhance effector T-cell infiltration into the tumors in preclinicalmodels. The major types of cancer vaccines include peptide vaccines,vector-based antigen specific vaccines, whole-cell vaccines, anddendritic cell vaccines. All vaccine-based therapies are designed todeliver either single or multiple antigenic epitopes or antigens fromthe whole cells to the patients and induce tumor-specific effector Tcells. Thus, a vaccine-based therapy may be the most efficient way toinduce T-cell infiltration into the tumor.

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host.

TIGIT, PD-1 and/or PD-L1 inhibition may be used in conjunction with acollection of recombinant proteins and/or peptides expressed in a tumorin order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self-antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). Tumor antigen can also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences (i.e., bcr-abl inthe Philadelphia chromosome), or idiotype from B cell tumors.

Non-limiting examples of tumor vaccines include sipuleucel-T(Provenge®), an FDA-approved tumor vaccine for Metastatic prostatecancer; tumor cells transfected to express the cytokine granulocytemacrophage colony-stimulating factor (GM-CSF), such as the whole cellGM-CSF-secreting irradiated, allogeneic pancreatic cancer vaccine (GVAX;Johns Hopkins); a multi-peptide vaccine consisting of immunogenicpeptides derived from breast cancer antigens, neu, legumain, and.beta.-catenin, which prolonged the vaccine-induced progression-freesurvival of breast tumor-bearing mice when administered in combinationwith anti-PD-1 antibody (Karyampudi L. et al. (2014) Cancer Res74:2974-2985); peptides of melanoma antigens, such as peptides of gp100,MAGE antigens, Trp-2, MARTI and/or tyrosinase, or. Other tumor vaccinesinclude proteins from viruses implicated in human cancers such as humanpapilloma viruses (HPV) (e.g., Gardasil®, Gardasil 9®, and Cervarix®;hepatitis B virus (e.g., Engerix-B and Recombivax HB); hepatitis C virus(HCV), Kaposi's sarcoma associated herpes sarcoma virus (KSHV). Anotherform of tumor specific antigen that can be used in conjunction withTIGIT inhibition is purified heat shock proteins (HSP) isolated from thetumor tissue itself. These heat shock proteins contain fragments ofproteins from the tumor cells and these HSPs are highly efficient atdelivery to antigen presenting cells for eliciting tumor immunity.Talimogene laherparepvec (T-VEC, or Imlygic®) is an FDA-approvedoncolytic virus for the treatment of some patients with metastaticmelanoma that cannot be surgically removed.

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens, as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization may be effectively combined withTIGIT blocking to activate (unleash) more potent anti-tumor responses.

TIGIT, PD-1 and/or PD-L1 inhibition can also be combined with standardcancer treatments (e.g., surgery, radiation, and chemotherapy). Inparticular, TIGIT, PD-1 and/or PD-L1 inhibition can be effectivelycombined with chemotherapeutic regimes. In these instances, it may bepossible to reduce the dose of chemotherapeutic reagent administered(Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of sucha combination is a checkpoint regulator antagonist in combination withdecarbazine for the treatment of melanoma. Another example of such acombination is a checkpoint regulator antagonist in combination withinterleukin-2 (IL-2) for the treatment of melanoma. For example, thescientific rationale behind the combined use of TIGIT, PD-1 and/or PD-L1inhibition and chemotherapy to promote cell death is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withTIGIT, PD-1 and/or PD-L1 inhibition through cell death are radiation,surgery, and hormone deprivation. Each of these protocols creates asource of tumor antigen in the host. Angiogenesis inhibitors can also becombined with TIGIT, PD-1 and/or PD-L1 inhibition. Inhibition ofangiogenesis leads to tumor cell death, which may feed tumor antigeninto host antigen presentation pathways.

The anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies,bispecific checkpoint regulator antagonists and trispecific checkpointregulator antagonists described herein may also be used in combinationwith bispecific antibodies that target Fcα or Fcγ receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would be augmentedby the inhibition of TIGIT, PD-1 and/or PD-L1. Alternatively, antigenmay be delivered directly to DCs by the use of bispecific antibodiesthat bind to tumor antigen and a dendritic cell specific cell surfacemarker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofimmunosuppressive proteins expressed by the tumors. These include amongothers TGF-.beta., IL-10, and Fas ligand. Antibodies to each of theseentities can be used in combination with the checkpoint regulatorantagonists described herein to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies that activate host immune responsiveness can be used incombination with the checkpoint regulator antagonists described herein.These include molecules on the surface of dendritic cells that activateDC function and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity (Ridge et al. (1998)Nature 393: 474-478) and can be used in conjunction with anti-TIGITantibodies. Activating antibodies to T cell costimulatory molecules,such as OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169),CD137/4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997), andICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide forincreased levels of T cell activation. In addition, inhibitors of otherimmune checkpoint regulators, may also be used in conjunction withcheckpoint regulator antagonists described herein, as further describedbelow.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, TIGIT inhibition may be used to increasethe effectiveness of the donor engrafted tumor specific T cells byreducing graft vs. tumor responses.

Ex vivo activation and expansion of antigen specific T cells andadoptive transfer of these cells into recipients in order to stimulateantigen-specific T cells against cancers or viral infections in thepresence of anti-TIGIT antibodies can increase the frequency andactivity of the adoptively transferred T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-TIGIT antibodies can increase the frequency andactivity of the adoptively transferred T cells.

In certain embodiments, a checkpoint regulator antagonist describedherein may be administered to a subject with an infectious disease,especially chronic infections. In this case, similar to its applicationto cancer, antibody-mediated TIGIT, PD-1 and/or PD-L1 inhibition can beused alone, or as an adjuvant, in combination with vaccines, to enhanceimmune responsiveness to pathogens, toxins, and self-antigens. Exemplarypathogens for which this therapeutic approach can be applied include,but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes,Giardia, Malaria, Leishmania, Staphylococcus aureus, and Pseudomonasaeruginosa. TIGIT, PD-1 and/or PD-L1 inhibition is particularly usefulagainst established infections by agents such as HIV that present novelor altered antigens over the course of the infections. Administration ofthe anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies,bispecific checkpoint regulator antagonists, and trispecific checkpointregulator antagonists can allow for recognition of these antigens asforeign so as to provoke an appropriate T cell response.

Other pathogenic viruses causing infections treatable by the methodsdescribed herein include HIV, hepatitis (A, B, or C), herpesvirusinfections (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barrvirus), and infections caused by an adenovirus, influenza virus,flavivirus, echoviruses, rhinoviruses, coxsackie viruses, coronaviruses,respiratory syncytial viruses, mumps viruses, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus,arboviral encephalitis virus, or combination thereof.

Exemplary pathogenic bacteria or diseases caused therefrom which may betreatable by the methods described herein include Chlamydia, Rickettsia,Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococciand Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella,Diphtheria, Salmonella, Bacilli, Cholera, Leptospirosis tetanus,botulism, anthrax, plague, and Lyme disease.

Exemplary pathogenic fungi causing infections treatable by the methodsdescribed herein include Candida (e.g., albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g.,fumigatus, niger, etc.), Mucorales (e.g., mucor, absidia, rhizopus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Exemplary pathogenic parasites causing infections treatable by themethods described herein include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia Zambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, TIGIT, PD-1 and/or PD-L1 inhibition can becombined with other forms of immunotherapy such as cytokine treatment(e.g., interferons, GM-CSF, G-CSF, IL-2), or bispecific antibody therapyusing two different binding specificities to provide enhancedpresentation of tumor antigens.

Anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies,bispecific checkpoint regulator antagonists, and trispecific checkpointregulator antagonists described herein can be used to enhanceantigen-specific immune responses by co-administration of one or more ofany of these antibodies with an antigen of interest (e.g., a vaccine).Accordingly, provided herein are methods of enhancing an immune responseto an antigen in a subject, comprising administering to the subject: (i)the antigen; and (ii) an anti-TIGIT antibody, anti-PD-1 antibody,anti-PD-L1 antibody, bispecific checkpoint regulator antagonist,trispecific checkpoint regulator antagonist, or combination thereof,such that an immune response to the antigen in the subject is enhanced.The antigen can be, for example, a tumor antigen, a viral antigen, abacterial antigen or an antigen from a pathogen. Non-limiting examplesof such antigens include those discussed in the sections above, such asthe tumor antigens (or tumor vaccines) discussed above, or antigens fromthe viruses, bacteria or other pathogens described above.

In certain embodiments, a peptide or fusion protein comprising theepitope to which an anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1antibody, bispecific checkpoint regulator antagonist, trispecificcheckpoint regulator antagonist binds may be used as a vaccine insteadof, or in addition to, the checkpoint regulator antagonist(s).

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multi-specific antibodies or antagonists andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

Combination Therapies

In another aspect, the present application provides combinationtherapies for enhancing an antigen-specific T cell response in asubject. In one embodiment, the method includes contacting a T cell withan anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody,antibody fragment thereof, bispecific checkpoint regulator antagonist,or trispecific checkpoint regulator antagonist in combination with asecond antibody, antibody fragment, antagonist or drug such that anantigen-specific T cell response or apoptotic pathway is enhanced. Forexample, in some embodiments, the first antibody or antibody fragmentspecifically binds TIGIT and the second antibody or antibody fragmentspecifically binds to PD-1 or PD-L1. In some embodiments, the secondantibody or antibody fragment comprises one or more anti-PD-1 CDRs ofFIG. 3B. In some embodiments, the second antibody or antibody fragmentcomprises one or more anti-PD-1 variable regions of FIGS. 4D-E.

In a related aspect, a method of reducing or depleting regulatory Tcells in a tumor of a subject in need thereof includes administering aneffective amount of an antibody or antibody fragment in combination witha second antibody, antibody fragment, antagonist or drug such that thenumber of regulatory T cells in the subject is reduced.

In some embodiments, the subject has a cell proliferative disease orcancer as described herein.

In other embodiments, the subject has a chronic viral infection,inflammatory disease or automimmune disease as described herein.

The provision of two distinct signals to T-cells is a widely acceptedmodel for lymphocyte activation of resting T lymphocytes byantigen-presenting cells (APCs). This model further provides for thediscrimination of self from non-self and immune tolerance. The primarysignal, or antigen specific signal, is transduced through the T-cellreceptor (TCR) following recognition of foreign antigen peptidepresented in the context of the major histocompatibility-complex (MHC).The second or co-stimulatory signal is delivered to T-cells byco-stimulatory molecules expressed on antigen-presenting cells (APCs),and induce T-cells to promote clonal expansion, cytokine secretion andeffector function. In the absence of co-stimulation, T-cells can becomerefractory to antigen stimulation, which results in a tolerogenicresponse to either foreign or endogenous antigens.

In the two-signal model, T-cells receive both positive co-stimulatoryand negative co-inhibitory signals. The regulation of such positive andnegative signals is critical to maximize the host's protective immuneresponses, while maintaining immune tolerance and preventingautoimmunity. Negative signals seem necessary for induction of T-celltolerance, while positive signals promote T-cell activation. Bothco-stimulatory and co-inhibitory signals are provided to antigen-exposedT cells, and the interplay between co-stimulatory and co-inhibitorysignals is essential to controlling the magnitude of an immune response.Further, the signals provided to the T cells change as an infection orimmune provocation is cleared, worsens, or persists, and these changespowerfully affect the responding T cells and re-shape the immuneresponse.

The mechanism of co-stimulation is of therapeutic interest because themanipulation of co-stimulatory signals has shown to provide a means toeither enhance or terminate cell-based immune response. Recently, it hasbeen discovered that T cell dysfunction or anergy can occur concurrentlywith an induced and sustained expression of immune checkpointregulators, such as programmed death 1 polypeptide (PD-1) and itsligands, PD-L1 and PD-L2. PD-L1 is overexpressed in many cancers and isoften associated with poor prognosis (Thompson R H et al., Cancer Res2006, 66(7):3381). Further, the majority of tumor infiltrating Tlymphocytes predominantly express PD-1, in contrast to T lymphocytes innormal tissues and peripheral blood T lymphocytes indicating thatup-regulation of PD-1 on tumor-reactive T cells can contribute toimpaired antitumor immune responses (Blood 2009 114(8): 1537). This maybe due to exploitation of PD-L1 signaling mediated by PD-L1 expressingtumor cells interacting with PD-1 expressing T cells to result inattenuation of T cell activation and evasion of immune surveillance.Inhibition of the PD-L1/PD-1 interaction provides a means to enhance Tcell immunity, including CD8+ T cell-mediated killing of cancer cellsand tumors. Similar enhancements to T cell immunity have been observedby inhibiting the binding of PD-L1 to the binding partner B7-1.Consequently, therapeutic targeting of PD-1 and other immune checkpointregulators are an area of intense interest.

Combining inhibition of TIGIT, PD-1 and/or PD-L1 signaling with othersignaling pathways deregulated in tumor cells can provide a means forenhance treatment efficacy. In recent years, a number of immunecheckpoint regulators in the form of receptors and their ligands havebeen identified. One important family of membrane-bound ligands thatbind to co-stimulatory or co-inhibitory receptors is the B7 family,which includes CTLA-4 and its ligands, B7-1 and B7-2; PD-1 and itsligands, PD-L1 (B7-H1) and PD-L2 (B7-DC); B7-H2 (ICOS-L), B7-H3, B7-H4,B7-H5 (VISTA), and B7-H6. Additional immune checkpoint antagonistsinclude, but are not limited to TIM-3 and its ligand, Galectin-9; LAG-3and its ligands, including liver sinusoidal endothelial cell lectin(LSECtin) and Galectin-3; CD122 and its CD122R ligand; CD70, B7H3, B andT lymphocyte attenuator (BTLA), and VISTA (Le Mercier et al. (2015)Front. Immunol., (6), Article 418). In addition, a number of checkpointregulator antagonists have been identified and tested in variousclinical and pre-clinical models and/or approved by the FDA (Kyi et al.,FEBS Letters, 588:368-376 (2014). The concept of inhibitory receptorblockade, also known as immune checkpoint blockade, has been validatedby virtue of e.g., the FDA approval of the PD-1 inhibitors, nivolumaband pembrolizumab, as well as the anti-CTLA-4 antibody, ipilimumab formetastatic melanoma.

An immune checkpoint antagonist modulates or interferes with theactivity of the immune checkpoint regulator so that, as a result of thebinding to the checkpoint regulator or its ligand, signaling through thecheckpoint regulator receptor is blocked or inhibited. By inhibitingthis signaling, immune-suppression can be reversed so that T cellimmunity against cancer cells can be re-established or enhanced. Incontrast, an immune checkpoint agonist (of e.g., a costimulatorymolecule) stimulates the activity of an immune checkpoint regulator sothat, as a result of the binding to the checkpoint regulator or itsligand, signaling through the checkpoint regulator receptor isstimulated. By stimulating this signaling, T cell immunity againstcancer cells can be re-established or enhanced.

Accordingly, in one embodiment, a method for stimulating an immuneresponse in a subject comprises administering to the subject ananti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, antibodyfragment(s) thereof (e.g., anti-TIGIT HCVR and/LCVRs) or bispecificcheckpoint regulator antagonist, or trispecific checkpoint regulatorantagonist described herein in combination with another immunecheckpoint regulator described herein above, such that an immuneresponse is stimulated in the subject, for example to inhibit tumorgrowth or to stimulate an anti-viral response.

In one embodiment, an anti-TIGIT antibody, anti-PD-1 antibody,anti-PD-L1 antibody, antibody fragment(s) thereof, bispecific checkpointregulator antagonist, or trispecific checkpoint regulator antagonistaccording to the present application is administered in combination withanother immune checkpoint regulator, either as separate antibodies or inmulti-specific antibody comprising binding specificities to multipleproducts. Generally, an anti-TIGIT antibody, anti-PD-1 antibody,anti-PD-L1 antibody, bispecific checkpoint regulator antagonist, ortrispecific checkpoint regulator antagonist described herein can becombined to stimulate an immune response with (i) an antagonist of theIgSF family protein, B7 family or TNF family that inhibit T cellactivation, or antagonist of a cytokine that inhibits T cell activation(e.g., IL-6, IL-10, TGF-.beta., VEGF, or other immunosuppressivecytokines) and/or (ii) an agonist of a stimulatory receptors of the IgSFfamily, B7 family or TNF family or of cytokines to stimulate T cellactivation, for stimulating an immune response.

In one embodiment, the subject is administered an anti-TIGIT antibody orHCVR and/or LCVR fragments thereof in combination with an anti-PD-1antibody or PD-1 antagonist. In another embodiment, the subject isadministered is administered an anti-TIGIT antibody or HCVR and/or LCVRfragments thereof in combination with an anti-PD-L1 antibody or PD-L1antagonist. In another embodiment, the subject is administered ananti-TIGIT antibody or HCVR and/or LCVR fragments thereof in combinationwith an anti-CTLA-4 antibody or CTLA-4 antagonist.

In certain embodiments, only subjects with a cancer exhibiting highexpression of a ligand for an immune checkpoint regulator are selectedfor combination treatment with the anti-TIGIT, anti-PD-1 and/oranti-PD-L1 antibody, fragment thereof, or any of the bispecific ortrispecific antagonists of the present application. By way of example,in one embodiment, a subject with a cancer exhibiting high expression ofPVR (CD155) and/or Nectin-2 (CD112) and/or low expression PD-L1 may beselected for monotherapy with anti-TIGIT antibodies, fragments thereof,or TIGIT antagonists of the present application, or combination therapywith a PD-1 antagonist or other immune checkpoint regulator.

The anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody may beadministered separately from the second antibody, antibody fragment orantagonist, or a multispecific antibody or antagonist may beadministered comprising at least one binding specificity for TIGIT and asecond binding specificity for the other targeted product. Further, theanti-TIGIT, anti-PD-1 antibody, anti-PD-L1 antibody or bispecific ortrispecific antagonist in accordance with the present application may beco-administered with one or more additional agents, e.g., antibodies,antagonists, or drugs in amount(s) effective in stimulating an immuneresponse and/or apoptosis so as to further enhance, stimulate orupregulate an immune response and/or apoptosis in a subject.

In some embodiments, the anti-TIGIT, anti-PD-1 or anti-PD-L1 antibody orfragment(s) thereof is administered subsequent to treatment with adifferent immune checkpoint regulator antagonist. For example, in oneembodiment, anti-TIGIT, anti-PD-1 or anti-PD-L1 antibodies may beadministered only after treatment with a PD-1/PD-L1 antagonist hasfailed, has led to incomplete therapeutic response, or there has beenrecurrence of the tumor or relapse (or “PD-1 failure”). In someembodiments, cancers exhibiting such failures may be screened forexpression of e.g., PVR and/or Nectin-2 and only those having high levelexpression are treated with an anti-TIGIT, anti-PD-1 or anti-PD-L1antibody, fragment or antagonist of the present application.

In a particular embodiment, the anti-TIGIT, anti-PD-1 or anti-PD-L1antibody or fragment is administered in combination with a PD-1, PD-L1,PD-L2 or TIGIT antagonist.

Other anti-PD-1 antibodies include, but are not limited to, nivolumab(BMS-936558, MDX-1106, OPDIVO™), a humanized immunoglobulin G4 (IgG4)mAb (Bristol-Myers Squibb); pembrolizumab (MK-3475, lambrolizumab,KEYTRUDA™)(Merck); pidilizumab (CT-011)(Medivation); and AMP-224(Merck). Anti-PD-1 antibodies are commercially available, for examplefrom ABCAM (AB137132), BIOLEGEND™ (EH12.2H7, RMP1-14) and AFFYMETRIXEBIOSCIENCE (J105, J116, MIH4). Anti-PD-1 antibodies also includesantibodies or antibody fragments containing one or more anti-PD-1 CDRsof FIG. 3B, or one or more anti-PD-1 variable regions of FIGS. 4D-E.

Other anti-PD-L1 antibodies include atezolizumab (MPDL3280A, RG7446), afully human IgG4 mAb Genentech/Roche); BMS-936559 (MDX-1105), a fullyhumanized IgG4 mAb (Bristol-Myers Squibb); MEDI4736, a humanized IgGantibody (Medimmune/AstraZeneca); and MSB0010718C, a fully human IgG4monoclonal antibody (Merck, EMD Serono).

Exemplary anti-CTLA-4 antibodies for use in accordance with the presentmethods include ipilimumab, trevilizumab and tremelimumab. Exemplaryanti-CTLA-4 dominant negative proteins include the humanized fusionprotein, Abatacept (Orencia), which comprises the Fc region of IgG1fused to the CTLA-4 ECD, and Belatacept (Nulojix®), a second generationhigher-affinity CTLA-4-Ig variant with two amino acid substitutions inthe CTLA-4 ECD relative to Abatacept.

In certain embodiments, the immune checkpoint regulator antagonist is adominant negative protein of the immune checkpoint regulator. Inparticular embodiments, the dominant negative protein comprises anextracellular domain derived from a member selected from the groupconsisting of PD-L1, PD-L2, PD-1, B7-1, B7-2, B7H3, CTLA-4, LAG-3,TIM-3, TIGIT, BTLA, VISTA, CD70, and combinations thereof. In certainparticular embodiments, these extracellular domains are fused to animmunoglobulin constant region or Fc receptor in the presently describedantibodies. Such mutants can bind to the endogenous receptor so as toform a complex that is deficient in signaling. In certain embodiments,the extracellular domain is fused to an immunoglobulin constant regionor Fc fragment or to a monomer in the oligomeric protein complex.

In certain embodiments, a dominant negative PD-L1 antagonist comprisesthe extracellular domain of PD-L1, PD-L2, or PD-1. In anotherembodiment, a dominant-negative PD-1 antagonist is employed, which has amutation so that it is no longer able to bind PD-L1. An exemplarydominant negative protein is AMP-224 (co-developed by Glaxo Smith Klineand Amplimmune), a recombinant fusion protein comprising theextracellular domain of PD-L2 and the Fc region of human IgG.

Exemplary immune checkpoint regulator agonists include, but are notlimited to members of the tumor necrosis factor (TNF) receptorsuperfamily, such as CD27, CD40, OX40, GITR and 4-1BB (CD137) and theirligands, or members of the B7-CD28 superfamily, including CD28 and ICOS(CD278). Additional checkpoint regulator agonists include CD2, CDS,ICAM-1, LFA-1 (CD11a/CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C,SLAMF7, NKp80, CD160, B7-H3, CD83 ligand. Immune checkpoint agonists caninclude antibodies or soluble fusion protein agonists comprising one ormore costimulatory domains. Agonist antibodies include, but are notlimited to anti-CD40 mAbs, such as CP-870,893, lucatumumab, anddacetuzumab; anti-CD137 mAbs, such as BMS-663513 urelumab, andPF-05082566; anti-OX40 mAbs; anti-GITR mAbs, such as TRX518; anti-CD27mAbs, such as CDX-1127; and anti-ICOS mAbs.

Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. Nos. 6,111,090 and8,586,023; European Patent No.: 090505B1, U.S. Pat. No. PCT PublicationNos.: WO 2010/003118 and 2011/090754. Anti-GITR antibodies are describedin, e.g., in U.S. Pat. Nos. 7,025,962, 7,618,632, 7,812,135, 8,388,967,and 8,591,886; European Patent Nos.: 1947183B1 and 1866339; PCTPublication Nos.: WO 2011/028683, WO 2013/039954, WO2005/007190, WO2007/133822, WO2005/055808, WO 99/40196, WO 2001/03720, WO99/20758,WO2006/083289, WO 2005/115451, WO 2011/051726. An exemplary anti-GITRantibody is TRX518.

Another family of membrane bound ligands that bind to co-stimulatory orco-inhibitory receptors is the TNF family of molecules that bind tocognate TNF receptor family members, which include CD40 and CD40L,OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137/4-1BB,TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,RANKL, TWEAKR/Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT3R,LIGHT, DcR3, HVEM, VEGI/TL 1A, TRAMP/DR3, EDAR, EDA, XEDAR, EDA2, TNFR1,Lymphotoxin α/TNFγ, TNFR2, TNFα, LT3R, Lymphotoxin α 1 (32, FAS, FASL,RELT, DR6, TROY, NGFR (see, e.g., Tansey, M. G. et al. (2009) DrugDiscovery Today, 14 (23-24):1082-1088).

Immune checkpoint agonists or costimulatory molecules include cellsurface molecules other than antigen receptors or their ligands that arerequired for an efficient immune response, and include, but are notlimited to MHC class I molecules, TNF receptor proteins,immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), activating NK cellreceptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30,CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS,ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7,NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

In one aspect, T cell responses can be stimulated by a combination ofthe anti-TIGIT, anti-PD-1 or anti-PD-L1 mAbs of the present inventionand one or more of (i) an antagonist of a protein that inhibits T cellactivation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1,PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69,Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-1,CD96 and TIM-4, and (ii) an agonist of a protein that stimulates T cellactivation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40,ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may becombined with anti-TIGIT antibodies, e.g., those described herein, fortreating cancer, include: YERVOY™/ipilimumab or tremelimumab (toCTLA-4), galiximab (to B7.1), OPDIVO™/nivolumab/BMS-936558 (to PD-1),pidilizumab/CT-011 (to PD-1), KEYTRUDA™/pembrolizumab/MK-3475 (to PD-1),AMP224 (to B7-DC/PD-L2), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1),MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (toLAG-3), urelumab/BMS-663513 and PF-05082566 (to CD137/4-1BB), CDX-1127(to CD27), MEDI-6383 and MEDI-6469 (to OX40), RG-7888 (to OX40L),Atacicept (to TACI), CP-870893 (to CD40), lucatumumab (to CD40),dacetuzumab (to CD40), and muromonab-CD3 (to CD3).

Other molecules that can be combined with the checkpoint regulatorantagonists described herein for the treatment of cancer includeantagonists of inhibitory receptors on NK cells or agonists ofactivating receptors on NK cells. For example, antagonist anti-TIGIT,anti-PD-1, and/or anti-PD-L1 antibodies can be combined with antagonistsof KIR (e.g., lirilumab), CSF-1R antagonists, such as RG7155.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofimmunosuppressive proteins expressed by the tumors. These include amongothers TGF-.beta., IL-10, and Fas ligand. Antibodies to each of theseentities can be used in combination with the checkpoint regulatorantagonists described herein to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies that activate host immune responsiveness can be used incombination with the checkpoint regulator antagonists described herein.These include molecules on the surface of dendritic cells that activateDC function and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity and can be used inconjunction with the checkpoint regulator antagonists described herein.Activating antibodies to T cell costimulatory molecules such as OX-40,CD137/4-1BB, and ICOS may also provide for increased levels of T cellactivation.

In certain embodiments, the checkpoint regulator antagonists describedherein can be co-administered with one or other more therapeutic agents,e.g., anti-cancer agents, radiotoxic agents or an immunosuppressiveagent. Such co-administration can solve problems due to development ofresistance to drugs, changes in the antigenicity of the tumor cells thatwould render them unreactive with the antibody, and toxicities (byadministering lower doses of one or more agents).

The checkpoint regulator antagonists described herein can be linked tothe agent (as an immuno-complex) or can be administered separate fromthe agent. In the latter case (separate administration), the antibodycan be administered before, after or concurrently with the agent or canbe co-administered with other known therapies, e.g., an anti-cancertherapy, e.g., radiation. The checkpoint regulator antagonists describedherein may be co-administered with one or more anti-cancer agents so asto provide two anti-cancer agents operating synergistically viadifferent mechanisms to yield a cytotoxic effect in human cancer cells.

The checkpoint regulator antagonists described herein may be combinedwith an anti-cancer agent, such an alkylating agent; an anthracyclineantibiotic; an anti-metabolite; a detoxifying agent; an interferon; apolyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor;a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; aphosphatidylinositol-3-kinase (PI3K) inhibitor; an Akt inhibitor; amammalian target of rapamycin (mTOR) inhibitor; a proteasomal inhibitor;a poly(ADP-ribose) polymerase (PARP) inhibitor; a Ras/MAPK pathwayinhibitor; a centrosome declustering agent; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitor; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase or a protein methyltransferase), a cytidine analogue orcombination thereof.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary anthracycline antibiotics include, but are not limited to,doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone(Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubidine);daunorubicin liposomal (DaunoXome); dactinomycin (Cosmegen); epirubicin(Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin(Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/odinel31 tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Exemplary histone deacetylase inhibitors include, but are not limitedto, vorinostat (Zolinza), valproic acid, romidepsin, entinostatabexinostat, givinostat, and mocetinostat.

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron);abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary phosphatidyl-inositol-3 kinase (PI3K) inhibitors includewortmannin an irreversible inhibitor of PI3K, demethoxyviridin aderivative of wortmannin, LY294002, a reversible inhibitor of PI3K;BKM120 (Buparlisib); Idelalisib (a PI3K Delta inhibitor); duvelisib(IPI-145, an inhibitor of PI3K delta and gamma); alpelisib (BYL719), analpha-specific PI3K inhibitor; TGR 1202 (previously known as RP5264), anoral PI3K delta inhibitor; and copanlisib (BAY 80-6946), an inhibitorPI3Kα,δ isoforms predominantly.

Exemplary Akt inhibitors include, but are not limited to miltefosine,AZD5363, GDC-0068, MK2206, Perifosine, RX-0201, PBI-05204, GSK2141795,and SR13668.

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus;deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128,BEZ235, PI-103, Torin1, PP242, PP30, Ku-0063794, WAY-600, WYE-687,WYE-354, and CC-223.

Exemplary proteasomal inhibitors include, but are not limited to,bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib(CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912),marizomib (NPI-0052), and disufiram.

Exemplary PARP inhibitors include, but are not limited to, olaparib,iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016,MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338 andcombinations thereof.

Exemplary Ras/MAPK pathway inhibitors include, but are not limited to,trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026,R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, andGDC-0973.

Exemplary centrosome declustering agents include, but are not limitedto, griseofulvin; noscapine, noscapine derivatives, such as brominatednoscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN),N-(3-brormobenzyl) noscapine, aminonoscapine and water-solublederivatives thereof; CW069; the phenanthridene-derived poly(ADP-ribose)polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furamide,N2-(2-thienylmethyl)-5-nitro-2-furamide, andN2-benzyl-5-nitro-2-furamide.

Exemplary multi-kinase inhibitors include, but are not limited to,regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080;Zd6474; PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol;seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412;bryostatin; KAI-9803; SF1126; VX-680; Azdl1152; Arry-142886 (AZD-6244);SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1Mpotassium oxonate) or lovastatin.

In certain embodiments, the anti-TIGIT antibodies or anti-TIGIT antibodyfragments described herein may be combined with one or more angiogenesisinhibitors. Angiogenesis, the development of new blood vessels frompre-existing vessels, is essential for tumor growth and metastasis.Angiogenesis inhibition presents a potentially valuable strategy fortreating diseases, such as cancer, in which progression (e.g.,metastasis) is dependent on neovascularization. Two importantangiogenesis pathways include the vascular endothelial growth factor(VEGF) pathway and the Tie2 pathway. The principal VEGF pathway ismediated by the transmembrane tyrosine kinase VEGF-R2. Various isoformsof VEGF, particularly VEGF-A, bind to VEGF-R2, resulting in dimerizationand activation through phosphorylation of various downstream tyrosinekinases. The Tie2 pathway is another angiogenesis pathway for whichtherapeutic antibodies and small molecule drugs have been developed. TheTie2 tyrosine kinase receptor activates angiogenesis in response tobinding by one its angiopoietin (Ang) ligands (i.e., Ang1, Ang2, Ang3(mouse) and Ang4). Inhibition of angiogenesis leads to tumor cell death,which may feed tumor antigen into host antigen presentation pathways.

In some embodiments, the subject is administered an anti-TIGIT antibodyor HCVR and/or LCVR fragments thereof in combination with one or moreVEGF binding antagonists and/or one or more Tie2 receptor bindingantagonists. A VEGF binding antagonist binds to VEGF-A or its receptorVEGFR-2 so that, as a result of the binding, activation of VEGFR-2 byVEGF-A is blocked or inhibited. A Tie2 receptor binding antagonist bindsto the Tie2 tyrosine kinase receptor or one of its angiopoietin (Ang)ligands (i.e., Ang-1, Ang-2, Ang-3 and Ang-4) so that, as a result ofthe binding, activation of the Tie2 receptor by one or more of itsligands is blocked or inhibited.

A preferred VEGF antibody antagonist is bevacizumab (AVASTIN™), ahumanized antibody. Bevacizumab comprises mutated human IgG1 frameworkregions and antigen-binding complementarity-determining regions from themurine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding ofhuman VEGF-A to VEGFR-2. Approximately 93% of the amino acid sequence ofbevacizumab, including most of the framework regions, is derived fromhuman IgG1, and about 7% of the sequence is derived from the murineantibody A4.6.1. Bevacizumab has a molecular mass of about 149,000Daltons and is glycosylated.

Additional anti-VEGF antibodies include ranibizumab (trade nameLucentis™), a monoclonal antibody fragment derived from the same parentmurine antibody as bevacizumab; the G6 or B20 series antibodies (e.g.,G6-23, G6-31, B20-4.1) described in U.S. Publication No. 2006/0280747,2007/0141065 and/or 2007/0020267, as well the antibodies described inU.S. Pat. Nos. 7,060,269, 6,884,879, 6,582,959, 6,703,020; 6,054,297;U.S. Patent Application Publication Nos. U.S. 2007/059312, U.S.2006/009360, U.S. 2005/0186208, U.S. 2003/0206899, U.S. 2003/0190317,and U.S. 2003/0203409.

An exemplary dominant negative anti-VEGF antagonist is Aflibercept, arecombinant fusion protein containing VEGF-A binding portions from theextracellular domains of human VEGF receptors 1 and 2 fused to the humanIgG1 Fc portion. Aflibercept acts as a soluble receptor decoy forVEGF-A.

An exemplary anti-VEGFR-2 antagonist is the humanized IgG1 monoclonalantibody, Ramucirumab, which binds to the extracellular domain ofVEGFR-2, thereby blocking its interaction with VEGF-A.

Exemplary small molecule antagonists of the VEGF pathway includemultikinase inhibitors of VEGFR-2, including sunitinib, sorafenib,cediranib, pazonpanib and nintedanib.

The Tie2 receptor binding antagonist binds to the Tie2 tyrosine kinasereceptor or one of its angiopoietin (Ang) ligands (i.e., Ang-1, Ang-2,Ang-3 and Ang-4) so that, as a result of the binding, activation of theTie2 receptor by one or more of its ligands is blocked or inhibited. Inone embodiment, the Tie2 receptor binding antagonist is an inhibitorypeptide. In a specific embodiment, the inhibitory peptide comprises theamino acid sequence in SEQ ID NO: 185, i.e.,AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE. In anotherembodiment, the Tie2 receptor binding antagonist comprises the peptideof SEQ ID NO: 93 fused to an Fc fragment, i.e.,

(SEQ ID NO: 186) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGAQQEECEWDPWTCHEMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE.

Other peptide inhibitors of Tie2 activation (including Ang-2 inhibitors)include A-11 (Compugen), which comprises the amino acid sequenceETFLSTNKLENQ (SEQ ID NO: 187); the CVX-060 peptide (Pfizer); the CVX-037peptide (Pfizer); and CGEN-25017 (Compugen). Additional peptideinhibitors of Tie2 activation are described in U.S. Pat. No. 7,138,370.

Antibody inhibitors of Tie2 activation (and/or angiopoietin-2) includeAMG-780 (Amgen), MEDI-3617 (Medlmmune/AstraZeneca), DX-2240(Dyax/Sanofi-Aventis), REGN-910 (Sanofi/Regeneron), RG7594 (Roche), LCO6(Roche), TAvi6 (Roche), AT-006 (Roche/Affitech). Additional Tie2receptor binding antibody antagonists and antibody binding sequencestherefrom are described in U.S. Pat. Nos. 7,521,053, 7,658,924, and8,030,025, as well as U.S. Patent Application Publication Nos.2013/0078248, 2013/0259859, and 2015/0197578.

Tie2 binding antagonists also include the small molecule inhibitors,CGI-1842 (CGI Pharmaceuticals), LP-590 (Locus Pharmaceuticals),ACTB-1003 (Act Biotech/Bayer AG), CEP-11981 (Cephalon/Teva), MGCD265(Methylgene), Regorafenib (Bayer), Cabozantinib/XL-184/BMS-907351(Exelixis), Foretnib (Exelixis), MGCD-265 (MethylGene Inc.).

In certain embodiments, the checkpoint regulator antagonists describedherein are administered at a subtherapeutic dose, another anti-immunecheckpoint regulator antibody or antagonist is administered at asubtherapeutic dose, the angiogenesis antagonist is administered at asubtherapeutic dose, or any antagonist in a combination thereof is eachadministered at a subtherapeutic dose.

In certain embodiments, TIGIT, PD-1 and/or PD-L1 inhibition is combinedwith standard cancer treatments (e.g., surgery, radiation, andchemotherapy). TIGIT, PD-1 and/or PD-L1 inhibition can be effectivelycombined with chemotherapeutic regimes. In these instances, it may bepossible to reduce the dose of chemotherapeutic reagent administered. Anexample of such a combination is an anti-TIGIT, anti-PD-1 or anti-PD-L1antibody in combination with decarbazine for the treatment of melanoma.Another example of such a combination is an anti-TIGIT, anti-PD-1 oranti-PD-L1 antibody in combination with interleukin-2 (IL-2) for thetreatment of melanoma. It is believed that the combined use of TIGIT,PD-1 and/or PD-L1 inhibition and chemotherapy can enhance apoptosis andincrease tumor antigen presentation for cytotoxic immunity. Othersynergistic combination therapies include TIGIT, PD-1 and/or PD-L1inhibition through cell death when used in combination with radiation,surgery or hormone deprivation. Each of these protocols creates a sourceof tumor antigen in the host.

In certain embodiment, the checkpoint regulator antagonists describedherein can also be used in multi-specific antagonists or in combinationwith bispecific antibodies targeting Fcα. or Fcγ receptor-expressingeffector cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti-tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tocancer cells or tumors. This targeting may more effectively activatetumor specific responses. The T cell arm of these responses would beaugmented by the inhibition of TIGIT, PD-1 and/or PD-L1. Alternatively,antigen may be delivered directly to DCs by the use of bispecificantibodies that bind to tumor antigen and a dendritic cell specific cellsurface marker.

Nucleic Acids and Host Cells for Expressing Checkpoint RegulatorAntagonist

In another aspect, the present application provides nucleic acidsencoding the checkpoint regulator antagonist of the present application,and expression vectors comprising such nucleic acids. In someembodiments, nucleic acids encodes an HCVR and/or LCVR fragment of anantibody or fragment in accordance with the embodiments describedherein, or any of the other antibodies and antibody fragments describedherein.

DNA encoding an antigen binding site in a monoclonal antibody can beisolated and sequenced from the hybridoma cells using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of themonoclonal antibodies). Alternatively, amino acid sequences fromimmunoglobulins of interest may be determined by direct proteinsequencing, and suitable encoding nucleotide sequences can be designedaccording to a universal codon table. In other cases, nucleotide andamino acid sequences of antigen binding sites or other immunoglobulinsequences, including constant regions, hinge regions and the like may beobtained from published sources well known in the art.

Expression vectors encoding a particular monospecific, bispecific ortrispecific checkpoint regulator antagonist may be used to synthesizethe checkpoint regulator antagonists of the present disclosure incultured cells in vitro or they may be directly administered to apatient to express the checkpoint regulator antagonist in vivo or exvivo. As used herein, an “expression vector” refers to a viral ornon-viral vector comprising a polynucleotide encoding one or morepolypeptide chains corresponding to the monospecific, bispecific ortrispecific checkpoint regulator antagonists of the present disclosurein a form suitable for expression from the polynucleotide(s) in a hostcell for antibody preparation purposes or for direct administration as atherapeutic agent.

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, a DNA for a presequence or signal peptide isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a signalpeptide, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers may be used in accordance with conventionalpractice.

Nucleic acid sequences for expressing the checkpoint regulatorantagonists typically include an amino terminal signal peptide sequence,which is removed from the mature protein. Since the signal peptidesequences can affect the levels of expression, the polynucleotides mayencode any one of a variety of different N-terminal signal peptidesequences. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like.

The above described “regulatory sequences” refer to DNA sequencesnecessary for the expression of an operably linked coding sequence inone or more host organisms. The term “regulatory sequences” is intendedto include promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells or those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). Expression vectors generally contain sequences fortranscriptional termination, and may additionally contain one or moreelements positively affecting mRNA stability.

The expression vector contains one or more transcriptional regulatoryelements, including promoters and/or enhancers, for directing theexpression of checkpoint regulator antagonists. A promoter comprises aDNA sequence that functions to initiate transcription from a relativelyfixed location in regard to the transcription start site. A promotercontains core elements required for basic interaction of RNA polymeraseand transcription factors, and may operate in conjunction with otherupstream elements and response elements.

As used herein, the term “promoter” is to be taken in its broadestcontext and includes transcriptional regulatory elements (TREs) fromgenomic genes or chimeric TREs therefrom, including the TATA box orinitiator element for accurate transcription initiation, with or withoutadditional TREs (i.e., upstream activating sequences, transcriptionfactor binding sites, enhancers, and silencers) which regulateactivation or repression of genes operably linked thereto in response todevelopmental and/or external stimuli, and trans-acting regulatoryproteins or nucleic acids. A promoter may contain a genomic fragment orit may contain a chimera of one or more TREs combined together.

Preferred promoters are those capable of directing high-level expressionin a target cell of interest. The promoters may include constitutivepromoters (e.g., HCMV, SV40, elongation factor-1.alpha. (EF-1.alpha.))or those exhibiting preferential expression in a particular cell type ofinterest. Enhancers generally refer to DNA sequences that function awayfrom the transcription start site and can be either 5′ or 3′ to thetranscription unit. Furthermore, enhancers can be within an intron aswell as within the coding sequence. They are usually between 10 and 300bp in length, and they function in cis. Enhancers function to increaseand/or regulate transcription from nearby promoters. Preferred enhancersare those directing high-level expression in the antibody producingcell. Cell or tissue-specific transcriptional regulatory elements (TREs)can be incorporated into expression vectors to restrict expression todesired cell types. Pol III promoters (H1 or U6) are particularly usefulfor expressing shRNAs from which siRNAs are expressed. An expressionvector may be designed to facilitate expression of the checkpointregulator antagonist in one or more cell types.

In certain embodiments, one or more expression vectors may be engineeredto express both the checkpoint regulator antagonist and one or moresiRNA targeting the Tie2 pathway, the VEGF pathway or an immunecheckpoint regulator.

An siRNA is a double-stranded RNA that can be engineered to inducesequence-specific post-transcriptional gene silencing of mRNAs.Synthetically produced siRNAs structurally mimic the types of siRNAsnormally processed in cells by the enzyme Dicer. When expressed from anexpression vector, the expression vector is engineered to transcribe ashort double-stranded hairpin-like RNA (shRNA) that is processed into atargeted siRNA inside the cell. Synthetic siRNAs and shRNAs may bedesigned using well known algorithms and synthesized using aconventional DNA/RNA synthesizer.

To co-express the individual chains of the checkpoint regulatorantagonist, a suitable splice donor and splice acceptor sequences may beincorporated for expressing both products. Alternatively, an internalribosome binding sequence (IRES) or a 2A peptide sequence, may beemployed for expressing multiple products from one promoter. An IRESprovides a structure to which the ribosome can bind that does not needto be at the 5′ end of the mRNA. It can therefore direct a ribosome toinitiate translation at a second initiation codon within a mRNA,allowing more than one polypeptide to be produced from a single mRNA. A2A peptide contains short sequences mediating co-translationalself-cleavage of the peptides upstream and downstream from the 2A site,allowing production of two different proteins from a single transcriptin equimolar amounts. CHYSEL is a non-limiting example of a 2A peptide,which causes a translating eukaryotic ribosome to release the growingpolypeptide chain that it is synthesizing without dissociating from themRNA. The ribosome continues translating, thereby producing a secondpolypeptide.

An expression vector may comprise a viral vector or a non-viral vector.A viral vectors may be derived from an adeno-associated virus (AAV),adenovirus, herpesvirus, vaccinia virus, poliovirus, poxvirus, aretrovirus (including a lentivirus, such as HIV-1 and HIV-2), Sindbisand other RNA viruses, alphavirus, astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,togaviruses and the like. A non-viral vector is simply a “naked”expression vector that is not packaged with virally derived components(e.g., capsids and/or envelopes).

In certain cases, these vectors may be engineered to target certaindiseases or cell populations by using the targeting characteristicsinherent to the virus vector or engineered into the virus vector.Specific cells may be “targeted” for delivery of polynucleotides, aswell as expression. Thus, the term “targeting”, in this case, may bebased on the use of endogenous or heterologous binding agents in theform of capsids, envelope proteins, antibodies for delivery to specificcells, the use of tissue-specific regulatory elements for restrictingexpression to specific subset(s) of cells, or both.

In some embodiments, expression of the antibody chains is under thecontrol of the regulatory element such as a tissue specific orubiquitous promoter. In some embodiments, a ubiquitous promoter such asa CMV promoter, CMV-chicken beta-actin hybrid (CAG) promoter, a tissuespecific or tumor-specific promoter to control the expression of aparticular antibody heavy or light chain or single-chain derivativetherefrom.

Non-viral expression vectors can be utilized for non-viral genetransfer, either by direct injection of naked DNA or by encapsulatingthe checkpoint regulator antagonist-encoding polynucleotides inliposomes, microparticles, microcapsules, virus-like particles, orerythrocyte ghosts. Such compositions can be further linked by chemicalconjugation to targeting domains to facilitate targeted delivery and/orentry of nucleic acids into desired cells of interest. In addition,plasmid vectors may be incubated with synthetic gene transfer moleculessuch as polymeric DNA-binding cations like polylysine, protamine, andalbumin, and linked to cell targeting ligands such as asialoorosomucoid,insulin, galactose, lactose or transferrin.

Alternatively, naked DNA may be employed. Uptake efficiency of naked DNAmay be improved by compaction or by using biodegradable latex beads.Such delivery may be improved further by treating the beads to increasehydrophobicity and thereby facilitate disruption of the endosome andrelease of the DNA into the cytoplasm.

Methods for Producing Monospecific or Multispecific Antibodies

In another aspect, the present application provides host cellstransformed with the anti-TIGIT, anti-PD-1 and/or anti-PD-L1 HCVRsand/or LCVRs encoding nucleic acids or expression vectors. The hostcells can be any bacterial or eukaryotic cell capable of expressing theanti-TIGIT, anti-PD-1 and/or anti-PD-L1 HCVRs and/or LCVRs encodingnucleic acids or expression vectors or any of the other co-administeredantibodies or antagonists described herein.

In another aspect, a method of producing a checkpoint regulatorantagonist comprises culturing a host cell transformed with one or moreanti-TIGIT, anti-PD-1 and/or anti-PD-L1 HCVRs and/or LCVRs encodingnucleic acids or expression vectors under conditions that allowsproduction of the antibody or fragment, and purifying the antibody fromthe cell.

In a further aspect, the present application provides a method forproducing an antibody comprising culturing a cell transiently or stablyexpressing one or more constructs encoding one or more polypeptidechains in the antibody; and purifying the antibody from the culturedcells. Any cell capable of producing a functional antibody may be used.In preferred embodiments, the antibody-expressing cell is of eukaryoticor mammalian origin, preferably a human cell. Cells from various tissuecell types may be used to express the antibodies. In other embodiments,the cell is a yeast cell, an insect cell or a bacterial cell.Preferably, the antibody-producing cell is stably transformed with avector expressing the antibody.

One or more expression vectors encoding the antibody heavy or lightchains can be introduced into a cell by any conventional method, such asby naked DNA technique, cationic lipid-mediated transfection,polymer-mediated transfection, peptide-mediated transfection,virus-mediated infection, physical or chemical agents or treatments,electroporation, etc. In addition, cells may be transfected with one ormore expression vectors for expressing the antibody along with aselectable marker facilitating selection of stably transformed clonesexpressing the antibody. The antibodies produced by such cells may becollected and/or purified according to techniques known in the art, suchas by centrifugation, chromatography, etc.

Examples of suitable selectable markers for mammalian cells includedihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycinanalog G418, hydromycin, and puromycin. When such selectable markers aresuccessfully transferred into a mammalian host cell, the transformedmammalian host cell can survive if placed under selective pressure.There are two widely used distinct categories of selective regimes. Thefirst category is based on a cell's metabolism and the use of a mutantcell line which lacks the ability to grow independent of a supplementedmedia. Two examples are CHO DHFR⁻ cells and mouse LTV cells. These cellslack the ability to grow without the addition of such nutrients asthymidine or hypoxanthine. Because these cells lack certain genesnecessary for a complete nucleotide synthesis pathway, they cannotsurvive unless the missing nucleotides are provided in a supplementedmedia. An alternative to supplementing the media is to introduce anintact DHFR or TK gene into cells lacking the respective genes, thusaltering their growth requirements. Individual cells which were nottransformed with the DHFR or TK gene will not be capable of survival innon-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, mycophenolic acid, orhygromycin. The three examples employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.Others include the neomycin analog G418 and puromycin.

Exemplary antibody-expressing cells include human Jurkat, humanembryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouseWEHI fibrosarcoma cells, as well as unicellular protozoan species, suchas Leishmania tarentolae. In addition, stably transformed, antibodyproducing cell lines may be produced using primary cells immortalizedwith c-myc or other immortalizing agents.

In one embodiment, the cell line comprises a stably transformedLeishmania cell line, such as Leishmania tarentolae. Leishmania areknown to provide a robust, fast-growing unicellular host for high levelexpression of eukaryotic proteins exhibiting mammalian-typeglycosylation patterns. A commercially available Leishmania eukaryoticexpression kit is available (Jena Bioscience GmbH, Jena, Germany).

In some embodiments, the cell lines expresses at least 1 mg, at least 2mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, or atleast 100 mg of the antibody/liter of culture.

The antibodies in the present application may be isolated from antibodyexpressing cells following culture and maintenance in any appropriateculture medium, such as RPMI, DMEM, and AIM V®. The antibodies can bepurified using conventional protein purification methodologies (e.g.,affinity purification, chromatography, etc.), including the use ofProtein-A or Protein-G immunoaffinity purification. In some embodiments,antibodies are engineered for secretion into culture supernatants forisolation therefrom.

Pharmaceutical Compositions and Methods of Treatment

Another aspect of the present application relates to pharmaceuticalcompositions and methods for treating a cell proliferative disorder,such as cancer, chronic infections, or immunologically compromiseddisease states. In one embodiment, the pharmaceutical compositioncomprises a checkpoint regulator antagonist of the present application.In some embodiments, the checkpoint regulator antagonist comprises ananti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, abispecific checkpoint regulator antagonist, a trispecific checkpointregulator antagonist, or an antigen-binding fragment thereof asdescribed herein, formulated together with a pharmaceutically acceptablecarrier. Such compositions may include one or more different antibodies,one or more multispecific antibodies, one or more immunoconjugates, or acombination thereof as described herein.

As described above, methods for using the pharmaceutical compositionsdescribed herein comprise administering to a subject in need thereof aneffective amount of the pharmaceutical composition according to thepresent disclosure.

Any suitable route or mode of administration can be employed forproviding the patient with a therapeutically or prophylacticallyeffective dose of the antibody or antagonist. Exemplary routes or modesof administration include parenteral (e.g., intravenous, intraarterial,intramuscular, subcutaneous, intratumoral), oral, topical (nasal,transdermal, intradermal or intraocular), mucosal (e.g., nasal,sublingual, buccal, rectal, vaginal), inhalation, intralymphatic,intraspinal, intracranial, intraperitoneal, intratracheal, intravesical,intrathecal, enteral, intrapulmonary, intralymphatic, intracavital,intraorbital, intracapsular and transurethral, as well as local deliveryby catheter or stent.

A pharmaceutical composition comprising an antibody or antagonist inaccordance with the present disclosure may be formulated in anypharmaceutically acceptable carrier(s) or excipient(s). As used herein,the term “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Pharmaceutical compositions may comprisesuitable solid or gel phase carriers or excipients. Exemplary carriersor excipients include but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols. Exemplary pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of thetherapeutic agents.

The checkpoint regulator antagonist can be incorporated into apharmaceutical composition suitable for parenteral administration.Suitable buffers include but are not limited to, sodium succinate,sodium citrate, sodium phosphate or potassium phosphate. Sodium chloridecan be used to modify the toxicity of the solution at a concentration of0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectantscan be included for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trehaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-Methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRIJ surfactants.

Therapeutic checkpoint regulator antagonist preparations can belyophilized and stored as sterile powders, preferably under vacuum, andthen reconstituted in bacteriostatic water (containing, for example,benzyl alcohol preservative) or in sterile water prior to injection.Pharmaceutical composition may be formulated for parenteraladministration by injection e.g., by bolus injection or continuousinfusion.

The therapeutic agents in the pharmaceutical compositions may beformulated in a “therapeutically effective amount” or a“prophylactically effective amount”. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the recombinant vector may varydepending on the condition to be treated, the severity and course of thecondition, the mode of administration, whether the antibody or agent isadministered for preventive or therapeutic purposes, the bioavailabilityof the particular agent(s), the ability of the checkpoint regulatorantagonist to elicit a desired response in the individual, previoustherapy, the age, weight and sex of the patient, the patient's clinicalhistory and response to the antibody, the type of the checkpointregulator antagonist used, discretion of the attending physician, etc. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the recombinant vector is outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result.

Preferably, the polypeptide domains in the checkpoint regulatorantagonist are derived from the same host in which they are to beadministered in order to reduce inflammatory responses against theadministered therapeutic agents.

The checkpoint regulator antagonist is suitably administered to thepatent at one time or over a series of treatments and may beadministered to the patient at any time from diagnosis onwards. Thecheckpoint regulator antagonist may be administered as the soletreatment or in conjunction with other drugs or therapies useful intreating the condition in question.

As a general proposition, a therapeutically effective amount orprophylactically effective amount of the checkpoint regulator antagonistwill be administered in a range from about 1 ng/kg body weight/day toabout 100 mg/kg body weight/day whether by one or more administrations.

In a particular embodiment, each checkpoint regulator antagonist isadministered in the range of from about 1 ng/kg body weight/day to about10 mg/kg body weight/day, about 1 ng/kg body weight/day to about 1 mg/kgbody weight/day, about 1 ng/kg body weight/day to about 100 μg/kg bodyweight/day, about 1 ng/kg body weight/day to about 10 μg/kg bodyweight/day, about 1 ng/kg body weight/day to about 1 μg/kg bodyweight/day, about 1 ng/kg body weight/day to about 100 ng/kg bodyweight/day, about 1 ng/kg body weight/day to about 10 ng/kg bodyweight/day, about 10 ng/kg body weight/day to about 100 mg/kg bodyweight/day, about 10 ng/kg body weight/day to about 10 mg/kg bodyweight/day, about 10 ng/kg body weight/day to about 1 mg/kg bodyweight/day, about 10 ng/kg body weight/day to about 100 μg/kg bodyweight/day, about 10 ng/kg body weight/day to about 10 μg/kg bodyweight/day, about 10 ng/kg body weight/day to about 1 μg/kg bodyweight/day, 10 ng/kg body weight/day to about 100 ng/kg body weight/day,about 100 ng/kg body weight/day to about 100 mg/kg body weight/day,about 100 ng/kg body weight/day to about 10 mg/kg body weight/day, about100 ng/kg body weight/day to about 1 mg/kg body weight/day, about 100ng/kg body weight/day to about 100 μg/kg body weight/day, about 100ng/kg body weight/day to about 10 μg/kg body weight/day, about 100 ng/kgbody weight/day to about 1 μg/kg body weight/day, about 1 μg/kg bodyweight/day to about 100 mg/kg body weight/day, about 1 μg/kg bodyweight/day to about 10 mg/kg body weight/day, about 1 μg/kg bodyweight/day to about 1 mg/kg body weight/day, about 1 μg/kg bodyweight/day to about 100 μg/kg body weight/day, about 1 μg/kg bodyweight/day to about 10 μg/kg body weight/day, about 10 μg/kg bodyweight/day to about 100 mg/kg body weight/day, about 10 μg/kg bodyweight/day to about 10 mg/kg body weight/day, about 10 μg/kg bodyweight/day to about 1 mg/kg body weight/day, about 10 μg/kg bodyweight/day to about 100 μg/kg body weight/day, about 100 μg/kg bodyweight/day to about 100 mg/kg body weight/day, about 100 μg/kg bodyweight/day to about 10 mg/kg body weight/day, about 100 μg/kg bodyweight/day to about 1 mg/kg body weight/day, about 1 mg/kg bodyweight/day to about 100 mg/kg body weight/day, about 1 mg/kg bodyweight/day to about 10 mg/kg body weight/day, about 10 mg/kg bodyweight/day to about 100 mg/kg body weight/day.

In other embodiments, the checkpoint regulator antagonist isadministered at a dose of 500 μg to 20 g every three days, or 25 mg/kgbody weight every three days.

In other embodiments, each checkpoint regulator antagonist isadministered in the range of about 10 ng to about 100 ng per individualadministration, about 10 ng to about 1 μg per individual administration,about 10 ng to about 10 μg per individual administration, about 10 ng toabout 100 μg per individual administration, about 10 ng to about 1 mgper individual administration, about 10 ng to about 10 mg per individualadministration, about 10 ng to about 100 mg per individualadministration, about 10 ng to about 1000 mg per injection, about 10 ngto about 10,000 mg per individual administration, about 100 ng to about1 μg per individual administration, about 100 ng to about 10 μg perindividual administration, about 100 ng to about 100 μg per individualadministration, about 100 ng to about 1 mg per individualadministration, about 100 ng to about 10 mg per individualadministration, about 100 ng to about 100 mg per individualadministration, about 100 ng to about 1000 mg per injection, about 100ng to about 10,000 mg per individual administration, about 1 μg to about10 μg per individual administration, about 1 μg to about 100 μg perindividual administration, about 1 μg to about 1 mg per individualadministration, about 1 μg to about 10 mg per individual administration,about 1 μg to about 100 mg per individual administration, about 1 μg toabout 1000 mg per injection, about 1 μg to about 10,000 mg perindividual administration, about 10 μg to about 100 μg per individualadministration, about 10 μg to about 1 mg per individual administration,about 10 μg to about 10 mg per individual administration, about 10 μg toabout 100 mg per individual administration, about 10 μg to about 1000 mgper injection, about 10 μg to about 10,000 mg per individualadministration, about 100 μg to about 1 mg per individualadministration, about 100 μg to about 10 mg per individualadministration, about 100 μg to about 100 mg per individualadministration, about 100 μg to about 1000 mg per injection, about 100μg to about 10,000 mg per individual administration, about 1 mg to about10 mg per individual administration, about 1 mg to about 100 mg perindividual administration, about 1 mg to about 1000 mg per injection,about 1 mg to about 10,000 mg per individual administration, about 10 mgto about 100 mg per individual administration, about 10 mg to about 1000mg per injection, about 10 mg to about 10,000 mg per individualadministration, about 100 mg to about 1000 mg per injection, about 100mg to about 10,000 mg per individual administration and about 1000 mg toabout 10,000 mg per individual administration. The checkpoint regulatorantagonist may be administered daily, every 2, 3, 4, 5, 6 or 7 days, orevery 1, 2, 3 or 4 weeks.

In other particular embodiments, the amount of the checkpoint regulatorantagonist may be administered at a dose of about 0.0006 mg/day, 0.001mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day or 10,000 mg/day. Asexpected, the dosage will be dependent on the condition, size, age andcondition of the patient.

In certain embodiments, the coding sequences for a checkpoint regulatorantagonist are incorporated into a suitable expression vector (e.g.,viral or non-viral vector) for expressing an effective amount of thecheckpoint regulator antagonist in patient with a cell proliferativedisorder. In certain embodiments comprising administration of e.g., oneor more recombinant AAV (rAAV) viruses, the pharmaceutical compositionmay comprise the rAAVs in an amount comprising at least 10¹⁰, at least10¹¹, at least 10¹², at least 10¹³, or at least 10¹⁴ genome copies (GC)or recombinant viral particles per kg, or any range thereof. In certainembodiments, the pharmaceutical composition comprises an effectiveamount of the recombinant virus, such as rAAV, in an amount comprisingat least 10¹⁰, at least 10¹, at least 10¹², at least 10¹³, at least10¹⁴, at least 10¹⁵ genome copies or recombinant viral particles genomecopies per subject, or any range thereof.

Dosages can be tested in several art-accepted animal models suitable forany particular cell proliferative disorder.

Delivery methodologies may also include the use of polycationiccondensed DNA linked or unlinked to killed viruses, ligand linked DNA,liposomes, eukaryotic cell delivery vehicles cells, deposition ofphotopolymerized hydrogel materials, use of a handheld gene transferparticle gun, ionizing radiation, nucleic charge neutralization orfusion with cell membranes, particle mediated gene transfer and thelike.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

EXAMPLES Example 1: Generation of Monoclonal Antibodies

Monoclonal antibodies (mAbs) of the present application are generatedand screened using techniques well known in the art, see, e.g., Harlowand Lane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York. The antigen specific hybridoma mabs are cloned,sequenced and engineered using techniques well known in the art, seee.g., Lo. B. K. C Methods in Molecular Biology™. Volume 248 2004.Antibody Engineering.

Example 2: TIGIT Binder Screening Assay

1 μg/ml TIGIT-His protein was coated overnight at 4° C. and blocked with1% BSA in PBS for 1 hour at room temperature and then incubated with 50μl hybridoma supernatant for 1 hour at room temperature, mouse IgG wasdetected by anti-mouse IgG HRP for 30 mins, TMB substrate was then addedand reaction was stopped by adding a 2N H2S04 solution. Wells with ODvalues at least five times over the background were selected as positivebinders.

Example 3: TIGIT Blocker Screening Assay

3×10⁴TIGIT+CHO-K1 cells were incubated in 50 ul hybridoma supernatantfor 20 mins at 4° C. and then human PVR human Fc tag fusion protein wasadded to a final concentration of 0.6 μ/ml; after 30 mins incubation at4° C., cells were washed with FACS buffer (0.5% BSA, 2 mM EMTA in PBS)and then cells were incubated with 1 μg/ml PE labeled anti-human Fcantibody for 20 mins at 4° C. Cells were then washed with FACS bufferand then re-suspended in 7-amino-actinomycin D (7AAD) solution beforeanalysis with iQue intellicyt system. Wells that completely blockedTIGIT and PVR (Fc tag) binding were selected as blockers. In the firstscreening, 18 blockers were identified out of 65 binders. In the secondscreening, 17 blockers were identified out of 30 binders. FIG. 1 showsexemplary results of a competition assay of various mouse anti-TIGITmabs.

Example 4: Anti-TIGIT Mab Blocking/ICS0 Assay

CHO-KI cells stably expressing human TIGIT were washed with FACS buffer(0.5% BSA, 2 mM EMTA in PBS) and resuspended at a concentration of 10 6cells/ml. Biotinylated hPVR-mIg (Cat #:555-030, Ancell) was added to theresuspended cells at 1 μg/ml; mixed well; without incubation, 20,000 ofthese CHO-KI cell (with human CD155/PVR protein) in 20 μl FACS buffer to96-well round bottom plates; 20 μl 2-fold serial dilutions of anti-humanTIGIT mab was added to the cells and incubated at 4 C for 30 min. Thecells were washed 2 times before staining with PE streptavidin(BioLegend) for 20 min at 4 C. The cells were then washed, resuspendedin 30 μl 7 AAD solution, and 35 μl of 10% neutral buffered formalinsolution was added. The cells were then incubated for 15 min. Flowcytometry was performed and IC50 was calculated using iQue intellicytsystem. Table 2 shows the IC50 of various anti-hu TIGIT mabs. Table 3shows binding constants for several anti-hu TIGIT mabs.

TABLE 2 Antibody IC50 (nM) T-01 0.29-0.56 T-02 0.35-0.71 T-03 0.26-0.52T-04 0.12-0.31 T-05 0.20-0.46 T-06 0.84 T-07 0.50 T-08 0.40-1.67 T-090.36-0.42 T-10 0.10-0.63

TABLE 3 Binding Affinity mAb K_(D) (nM) K_(a) (M⁻¹ s⁻¹) Kd (s⁻¹) T-102.01 8.56E+05 1.72E−03 T-04 0.67 8.09E+05 5.38E−04 T-08 5.44 6.90E+053.76E−03 BM 22 9.71E+05 2.13E−02

Example 5: Anti-TIGIT Mabs Binding Assay

FIG. 6 shows the binding of anti-TIGIT mouse mabs to cells expressingfull-length human TIGIT (hu TIGIT). Briefly, serial dilutions ofanti-TIGIT mabs were added to CHO-K1 cells (20,000 cells/well)overexpressing human or cyno TIGIT. The mixtures were incubated at 4 Cfor 20 min and washed 3 times. The mixtures were stained with thesecondary antibody, PE labeled F(ab′)2-Goat anti-human IgG Fc (ThermoH10104) at 4° C. for 20 min. Cells were washed and resuspended in 7AADsolution and fixed in 10% neutral buffered formalin solution for 15minutes before analysis with the iQue Intellicyt system.

Example 6: Anti-TIGIT Mabs Block the Interaction Between Hu TIGIT andits Ligand

Using the cell-based blocking assay described in Example 4, anti-TIGITmabs were evaluated for their ability to block the interaction betweenhu TIGIT and its ligand, human PVR (CD155). The results of this assayare shown in FIG. 7 .

Example 7: Human T Cell Assay with PBMC from Normal Donor and PVR Signalfrom Pre-Coated PVR-mIgG

Two human T cell assays were performed to show the functionality of theanti-TIGIT antibodies. Briefly, normal healthy human PBMC were activatedwith SEB (Toxin Technology, Cat #: BT202) or anti-CD3+Anti-CD28 for 5 to7 days in 6-well plates with PBMCs expressing high levels of TIGIT.96-well flat-bottom plates were coated with huPVR/CD155Mouse IgG2a FcTag (ACRO Biosystems) overnight at 4° C.

To assay for IFN-γ production, 100,000 cells were plated into thepre-coated wells and re-stimulated with 0.5 ug/ml Staphylococcalenterotoxin B (SEB). 66 μg/ml of anti-TIGIT mAbs or isotype control Abwas added. 3 to 5 days later, the supernatant was examined for IFN-γ byELISA. FIG. 8 shows that the anti-TIGIT mabs induced IFN-γ productionfrom the human PBMCs.

To assay for T cell proliferation, CellTrace Far Red labeled 100,000cells were plated into the pre-coated wells and re-stimulated with 0.5ug/ml SEB. 66 ug/ml of anti-TIGIT or an isotype-matched control antibodywere added into wells with coated human CD155/PVR. Total volume per wellwas 200 μl. The plates were incubated at 37° C. At 4 or 5 days poststimulation, PBMC cells were stained with FITC labeled anti-human CD3 Ab(BioLegend) and analyzed using the iQue intellicyt system. T cellproliferation indices were calculated based on the reduction of MFI(Mean Fluorescence Intensity) of CellTrace Far Red signal on gated CD3+T cells. As shown in FIG. 9 , the results from this assay showed thatthe anti-TIGIT mabs induced the proliferation of T cells.

Example 8: Binding of Anti-TIGIT Mabs to Hu TIGIT- and Cyno-Hu TIGIT

FIG. 10A shows binding of anti-TIGIT mabs to hu TIGIT- and cyno-hu TIGITusing the binding assay described in Example 5. FIG. 10B showsinhibition of antibody binding to hu TIGIT- and cyno-hu TIGIT byanti-TIGIT mabs using the blocking assay described in Example 4.

Example 9: PD-1 Binder Screening Assay

1 μg/ml huPD-1-His protein was coated in wells overnight at 4° C. andblocked with 1% BSA in PBS for 1 hour at room temperature and thenincubated with 50 μl hybridoma supernatant for 1 hour at roomtemperature. Mouse IgG was detected by incubating the wells withanti-mouse IgG HRP for 30 mins, adding TMB substrate, and stopping thereaction with 2N sulfuric acid solution. Wells with OD450 value at leastfive times over the background were selected as positive binders.

Example 10: PD-1 Blocker Screening Assay

3×10⁴ huPD1+CHO-K1 cells were incubated in 50 μl hybridoma supernatantfor 20 mins at 4° C. and then human PD-L1 human Fc tag fusion proteinwas added to a final concentration of 0.6 μg/ml. After a 30 minincubation at 4° C., the cells were washed with FACS buffer (0.5% BSA, 2mM EMTA in PBS) and then incubated with 1 μg/ml PE labeled anti-human Fcantibody for 20 mins at 4° C. Cells were then washed with FACS bufferand then re-suspended in 7AAD solution before analysis with iQueintellicyt system. Wells that completely blocked PD-1 and PD-L1 (Fc tag)binding were selected as blockers.

Example 11: Anti-PD-1Mab Blocking/IC50 Assay

A blocking assay was carried out to calculate the IC50 for selectedanti-PD-1 antibodies. Briefly, 2 or 3 fold serial dilutions ofanti-human PD-1 mAb or bispecific Ab (Highest Ab concentration: 128 nM;Triplicates for each mAb) were prepared. Human or Cyno PD-1 transfectedCHO-K1 cells were washed with FACS buffer (0.5% BSA 2 mM EDTA in PBS)and re-suspended at a concentration of 10⁶ cells/ml. FITC labeled humanPD-L-Fc protein was added to the human or cyno PD-1 transfected CHO-K1cells at a final concentration of 7 μg/ml and mixed well. Withoutincubation, 2,000 of these CHO-K1 cells (with PD-L1 Protein) in 20 μlFACS buffer was immediately added to a 96-well round bottom plate and 20μl or 2 or 3 fold serial diluted anti-human PD-1 mAbs were immediatelyadded to the cells and incubated at 4° C. for 30 mins. The cells werethen washed and re-suspended in 30 μl 7 AAD solution; 35 μl 10% neutralbuffered formalin solution was then added and incubated for 15 mins.before analysis using the iQue intellicyt system. FIGS. 11A, 12A and 12Bshow the results demonstrating the ability of several anti-PD-1 mabs toblock the interaction between human PD-1 and human PD-L1. FIG. 11B showsthe ability of anti-PD-1 mabs to block the interaction between cyno huPD-1 and cyno PD-L1.

Example 12: Anti-PD-1 Antibody/Kinetic Analysis by Bio-LayerInterferometry

Bio-light interferometry was carried out using the Octet RED96 system(ForteBio Octet RED96 System) to characterize the binding kinetics ofantibodies against His tagged human PD-1 protein. 20 nM of antibody wasloaded onto the anti-human IgG capture biosensors. Association ofanalyte (His tagged human PD-1 protein) was observed by placing thebiosensors in wells containing 2 or 3 fold serial dilution of analytes(72 nM being the highest concentration) for 5 mins. Dissociation wasmeasured after transfer of the biosensors into kinetic buffer alone andmonitoring of the interferometry signal for 10 minutes. The observed onand off rates (Ka and Kd) were fit using a 1:1 binding global fit modelcomprising at least 5 concentrations tested, and the equilibrium bindingconstant K_(D) was then calculated. FIGS. 13A-C shows the bindingaffinity of PD-01 mab (FIG. 13A), PD-02 mab (FIG. 13B), and benchmark(BM) anti-PD-1 mab (FIG. 13C).

Example 13: Enhanced IFN-γ Production by Anti-PD-1Mabs Alone or inCombination with Anti-TIGIT Mab

To show the ability of the anti-PD-1 antibodies to induce IFN-γproduction, 100,000 normal healthy human PBMC were activated with SEB(Toxin Technology). H358 cancer cell lines were added to provide PD-L1ligand signals. 10 μg/ml of anti-PD-1 mAbs or isotype control Ab wereadded. 3 days later, supernatant were examined for IFN-γ by ELISA. FIG.14 shows the enhancement of IFN-γ production from human PBMCs by theanti-PD-1 mabs depicted. FIGS. 15 AND 16B show that a combination ofanti-TIGIT (T-08) and anti-PD-1 (PD-01) mabs further increased IFN-γproduction from human PBMCs over the level of the anti-PD-1 antibodyalone.

Example 14: Enhanced T Cell Proliferation by Anti-PD-1Mabs Alone or inCombination with Anti-TIGIT Mab

To evaluate the ability of the anti-PD-1 antibodies to induce T cellproliferation, an assay was carried out similar to the assay describedin Example 7, except that H358 cancer cell lines were added to providePD-L1 ligand signals. FIGS. 16A and 16B show increased proliferation ofhuman T cell proliferation by a combination of anti-TIGIT- and anti-PD-1mabs.

Example 15: Screening Assays for Selection of Bispecific Antibodies

FIGS. 19A-D shows the results of screening assays to identify bispecificantibody configurations capable of blocking both PD-1 binding (FIG. 19A,FIG. 19B) and TIGIT binding (FIG. 19C, FIG. 19D). These screening assaysemployed the IC50 blocking assays described in Example 6 and 11.Antibody configurations exhibiting effective binding are shown in FIG.17 .

Example 16: Expression and Functional Evaluation of BispecificAntibodies

The blocking assay in Example 11 was used to calculate the IC50 forthree exemplary bispecific checkpoint regulator antagonists, TP-83,TP-92 and TP-93. FIGS. 20A-B shows the ability of these bispecificcheckpoint regulator antagonists to block binding of PD-L1 to PD-1 (FIG.20A) and block binding of PVR to TIGIT (FIG. 20B). These figures furthershow the calculated IC50s for blocking the binding of PD-L1 to PD-1(FIG. 20A) and for blocking the binding of PVR to TIGIT (FIG. 20B). FIG.21 shows a Coomasie stained gel showing production of bispecificantibodies, TP-83, TP-92 and TP-93.

Example 17: Exemplary Size Exclusion Chromatography (SEC) Profile ofBispecific Checkpoint Regulator Antagonists

FIG. 22 shows an exemplary SEC profile for exemplary bispecificcheckpoint regulator antagonists after one step purification. Briefly, a20 gig sample (either TP-93 or TP-83) was loaded onto a Zenix SEC-300column (3 μm, 300 .ANG., 7.8×300 mm). The mobile phase (150 mM sodiumphosphate, pH 7.0) was delivered at a flow rate of 0.80 mL/min. UVabsorbance was monitored at 280 nm. The main peaks represent the desiredproducts (86% for TP-93 and 74% for TP-83). The minor peaks representeither incorrectly aggregated or assembled products. This analysisunderscores the manufacturability of the bispecific checkpoint regulatorantagonists according to the present application.

Example 18: Increased Up-Regulation of IFN-γ Production by BispecificCheckpoint Regulator Antagonists

To evaluate the ability of a bispecific checkpoint regulator antagonist(TP-93) to induce T cell proliferation as compared to monospecificanti-TIGIT (T-08) and anti-PD-1 (PD-01) mabs, a CMV antigen specificrecall assay was performed. Briefly, PBMCs from individual donorspre-screened for CMV antigen reactivity (Donor 333 and Donor 287) werepurchased from Astarte Biologics. Cell lysates from CMV-infected cells(used as CMV antigen) were also purchased from Astarte Biologics. 1) ForIFN-g production: 250,000 PBMCs were plated and the antigen-specificstimulation was performed by the addition of 0.1 μg/ml of CMV Ag, whichstimulates the CMV reactive T cells. SHP-77 cells were co-cultured withPBMC to provide an immune function inhibitory environment. IgG4 control,anti-TIGIT Ab, anti-PD-1 Ab, anti-TIGIT Ab plus anti-PD-1 Ab, andbispecific Ab were separately added to each plate. 5 days later,supernatants were examined for IFN-γ production by ELISA. 2) For T-cellproliferation, 250,000 PBMCs were plated and activated with 0.1 μg/ml ofCMV Ag for 2 days and then labeled with CellTrace Far Red. 100,000labeled PBMC were then re-plated and re-stimulated with 1 μg/ml CMV Ag.128 nM of IgG4 control, anti-TIGIT Ab, the anti-PD-1 Ab, anti-TIGIT Abplus anti-PD-1 Ab, or bispecific Ab were separately added to each plate.Total volume per well was 200 μl. The plates were incubated at 37° C. or5 days post-stimulation and the PBMC cells were stained with PE labeledanti-human CD4 Ab (BioLegend) and analyzed by the iQue intellicytsystem. T cell proliferation indices were calculated based on thereduction of mean fluorescence intensity (MFI) of CellTrace Far Redsignal on gated CD4+ T cells. 3) T cell surface expression of TIGIT andPD-1 was also examined by FACS at day 5 with APC labeled anti-human CD3,PE labeled anti-TIGIT and FITC labeled anti-PD-1 Abs (data not shown).

FIGS. 23A and 23B show that the bispecific checkpoint regulatorantagonist, TP-93, increased IFN-γ production to a greater extent thanthe monospecific anti-TIGIT (T-08) and anti-PD-1 (PD-01) mabs alone or acombination of the monospecific anti-TIGIT and anti-PD-1 antibodiesusing two different human PBMC cell populations (Donor 333, FIG. 23A;Donor 287, FIG. 23B). FIGS. 23C and 23D show that the bispecificcheckpoint regulator antagonist, TP-93, exhibited a higher T cellproliferation index than the monospecific anti-TIGIT (T-08) andanti-PD-1 (PD-01) mabs alone or a combination of the monospecificanti-TIGIT and anti-PD-1 antibodies using two different human PBMC cellpopulations (Donor 333, FIG. 23C; Donor 287, FIG. 23D).

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. An antibody, or an antigen-binding portionthereof, comprising: (1) a heavy chain variable region, wherein theheavy chain variable region comprises three complementarity determiningregions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acidsequence selected from the group consisting of SEQ ID NOS:48, 51, 54, 56and 59, wherein HCDR2 has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:49, 52, 57 and 60, and wherein HCDR3 has anamino acid sequence selected from the group consisting of SEQ ID NOS:50,53, 55, 58 and 61; (2) a light chain variable region, wherein the lightchain variable region comprises three complementarity determiningregions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acidsequence selected from the group consisting of SEQ ID NOS:62, 65, 68,69, 70 and 73, wherein LCDR2 has an amino acid sequence selected fromthe group consisting of SEQ ID NOS:63, 66, 71 and 74, and wherein LCDR3has an amino acid sequence selected from the group consisting of SEQ IDNOS:64, 67, 72 and 75, wherein the antibody, or the antigen-bindingportion thereof, binds specifically to human PD-1.
 2. A bispecificcheckpoint regulator antagonist, comprising: an antigen binding domaincomprising the antigen-binding portion of the antibody of claim
 1. 3.The bispecific checkpoint regulator antagonist of claim 2, furthercomprising an antigen binding domain that binds specifically to humanTIGIT.
 4. The bispecific checkpoint regulator antagonist of claim 3,wherein the antigen binding domain that binds specifically to human TWATcomprises: (1) a heavy chain variable region, wherein the heavy chainvariable region comprises three complementarity determining regions(HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acidsequence selected from the group consisting of SEQ ID NOS:1, 6, 11, 15,17, 20 and 23, wherein HCDR2 has an amino acid sequence selected fromthe group consisting of SEQ ID NOS:2, 4, 7, 9, 12, 13, 16, 18, 21 and24, and wherein HCDR3 has an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 3, 5, 8, 10, 14, 19, 22 and 25; and (2) alight chain variable region, wherein the light chain variable regioncomprises three complementarity determining regions (LCDRs): LCDR1,LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence selected fromthe group consisting of SEQ ID NOS:26, 29, 31, 33, 35, 39, 42 and 45,wherein LCDR2 has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:27, 30, 36, 37, 40, 43 and 46, and whereinLCDR3 has an amino acid sequence selected from the group consisting ofSEQ ID NOS:28, 32, 34, 38, 41, 44 and
 47. 5. The bispecific checkpointregulator antagonist of claim 4, wherein the antigen binding domain thatbinds specifically to human TIGIT comprises an HCDR1 comprising theamino acid sequence of SEQ ID NO:23, an HCDR2 comprising the amino acidsequence of SEQ ID NO:24, an HCDR3 comprising the amino acid sequence ofSEQ ID NO:25, an LCDR1 comprising the amino acid sequence of SEQ IDNO:45, an LCDR2 comprising the amino acid sequence of SEQ ID NO:46, andan LCDR3 comprising the amino acid sequence of SEQ ID NO:47.
 6. Thebispecific checkpoint regulator antagonist of claim 5, wherein theantigen binding domain that binds specifically to human TIGIT comprisesa heavy chain variable region comprising the amino acid sequence of SEQID NO:125 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:126.
 7. A method for treating a cell proliferativedisorder in a subject, comprising: administering to a subject in needthereof an effective amount of the bispecific checkpoint regulatorantagonist of claim
 2. 8. The antibody, or an antigen-binding portionthereof, of claim 1, wherein the HCDR1 comprises the amino acid sequenceof SEQ ID NO:56, the HCDR2 comprises the amino acid sequence of SEQ IDNO:57, the HCDR3 comprises the amino acid sequence of SEQ ID NO:58, theLCDR1 comprises the amino acid sequence of SEQ ID NO:70, the LCDR2comprises the amino acid sequence of SEQ ID NO:71, and the LCDR3comprises the amino acid sequence of SEQ ID NO:
 72. 9. The antibody, oran antigen-binding portion thereof, of claim 1, wherein the heavy chainvariable region comprises the amino acid sequence of SEQ. ID NO:135 andwherein the light chain variable region comprises the amino acidsequence of SEQ ID NO:136.
 10. A bispecific checkpoint regulatorantagonist, comprising: an antigen binding domain comprising theantigen-binding portion of the antibody of claim
 8. 11. The bispecificcheckpoint regulator antagonist of claim 10, further comprising anantigen binding domain that binds specifically to human TIGIT.
 12. Thebispecific checkpoint regulator antagonist of claim 11, wherein theantigen binding domain that binds specifically to human TIGIT comprises:(1) a heavy chain variable region, wherein the heavy chain variableregion comprises three complementarity determining regions (HCDRs):HCDR1, HCDR2 and HCDR3, wherein HCDR1has an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 1, 6, 11, 15, 17, 20 and 23,wherein HCDR2 has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:2, 4, 7, 9, 12, 13, 16, 18, 21 and 24, andwherein HCDR3 has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:3, 5, 8, 10, 14, 19, 22 and 25; and (2) a lightchain variable region, wherein the light chain variable region comprisesthree complementarity determining regions (LCDRs): LCDR1, LCDR2 andLCDR3, wherein LCDR1 has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:26, 29, 31, 33, 35, 39, 42 and 45, wherein LCDhas an amino acid sequence selected from the group consisting of SEQ IDNOS:27, 30, 36, 37, 40, 43 and 46, and wherein LCDR3 has an amino acidsequence selected from the group consisting of SEQ ID NOS:28, 32, 34,38, 41, 44 and
 47. 13. The bispecific checkpoint regulator antagonist ofclaim 12, wherein the antigen binding domain that binds specifically tohuman TIGIT comprises an HCDR1 comprising the amino acid sequence of SEQID NO:23, an HCDR2 comprising the amino acid sequence of SEQ ID NO:24,an HCDR3 comprising the amino acid sequence of SEQ ID NO:25, an LCDR1comprising the amino acid sequence of SEQ ID NO:45, an LCDR2 comprisingthe amino acid sequence of SEQ ID NO:46, and an LCDR3 comprising theamino acid sequence of SEQ ID NO:47.
 14. The bispecific checkpointregulator antagonist of claim 13, wherein the antigen binding domainthat binds specifically to human TIGIT comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:125 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:126.
 15. A method for treating a cell proliferative disorder in asubject, comprising: administering to a subject in need thereof aneffective amount of the bispecific checkpoint regulator antagonist ofclaim
 10. 16. An antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that comprises the amino acidsequence of SEQ ID NO:137 and a light chain variable region thatcomprises the ammo acid sequence of SEQ ID NO:138, wherein the antibody,or an antigen-binding portion thereof, binds to human PD-1.
 17. Theantibody, or an antigen-binding portion thereof, of claim 1, wherein theHCDR1 comprises the amino acid sequence of SEQ ID NO:59, the HCDR2comprises the amino acid sequence of SEQ ID NO:60, the HCDR3 comprisesthe amino acid sequence of SEQ ID NO:61, the LCDR1 comprises the aminoacid sequence of SEQ ID NO:73, the LCDR2 comprises the amino acidsequence of SEQ ID NO:74, and the LCDR3 comprises the amino acidsequence of SEQ ID NO:75.